Identification of D-threo-alpha-methylisocitrate as stereochemically specific substrate for bovine heart aconitase and inhibitor of TPN-linked isocitrate dehydrogenase.

DL-threo-alpha-Methylisocitrate (3-hydroxy-1,2,3-butanetricarboxylate) is a substrate for bovine heart aconitase and an inhibitor of TPN-linked isocitrate dehydrogenase from liver and heart. The isomer of alpha-methylisocitrate formed from alpha-methyl-cis-aconitate (cis-2-butane-1,2,3-tricarboxylate) by aconitase inhibits TPN-linked isocitrate dehydrogenase and has been identified as D-threo-alpha-methylisocitrate (2S,3R)-3-hydroxy-1,2,3-butanetricarboxylate) by optical rotation and circular dichroism studies. Mitochondrial bovine heart aconitase catalyzes a reversible reaction between D-threo-alpha-methylisocitrate (Km, 0.2 mM) and alpha-methyl-cis-aconitate (Km, 0.05 mM) at pH 7.4. However, formation of methylcitrate (2-hydroxy-1,2,3-butanetricarboxylate) from these substrates or utilization of synthetic methylcitrate for formation of these products could not be demonstrated with bovine heart aconitase. DL-threo-alpha-Methylisocitrate is also a substrate for aconitase from rat liver cytosol (Km, 0.1 mM); Vmax with citrate is approximately 1.4 times that with DL-threo-alpha-methylisocitrate. The ratio of activities for these substrates observed with the bovine heart enzyme is about 5. Formation of alpha-methyl-cis-aconitate from synthetic methylcitrate could not be detected spectrophotometrically with the liver aconitase; if it occurs with either the liver or the heart enzyme, the rate would be less than 0.1% that obtained with DL-threo-alpha-methylisocitrate. A new synthesis of methylcitric acid in good yields from diethyl alpha-methyl-beta-ketoglutarate (diethyl 2-methyl-3-oxoglutarate) and cyanide has been described. NMR spectroscopy indicates that this synthetic methylcitric acid contains the two racemic pairs of diastereoisomers.


Lipoprotein regulation of 3-hydroxy-3-methylglutaryl coenzyme A reductase activity in rat liver cell cultures.

A primary cell culture technique was used to study the effects of lipoproteins on rat hepatocyte 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase activity. In this system, lipoproteins prepared from normocholesterolemic rat and human plasma, including low density lipoproteins, did not inhibit hepatocyte HMG-CoA reductase activity whereas very low density lipoproteins and high density lipoproteins isolated from the same sources were stimulatory. A lipoprotein was isolated from the plasma of cholesterol-fed rats that did inhibit hepatocyte HMG-CoA reductase activity. An extensive chemical characterization of the inhibitory lipoprotein revealed that it was mainly d less than 1.019 g/ml and had beta mobility on lipoprotein electrophoresis. The lipoprotein was compared to the comparable density fraction in the normocholesterolemic rat plasma and there was no size difference appreciable by negatively stained electron micrographs. However, two important differences in chemical composition were evident: in the inhibitory lipoproteins the per cent of total apoprotein which was in the region of Mr = 35,000 was increased 1.5- to 2-fold, and there was a marked increase in cholesterol ester content. These chemical characteristics may be required for lipoproteins to regulate hepatocyte cholesterol synthesis. Primary cell culture of rat hepatocytes appears to be a useful system in which to study cholesterol metabolism in the liver.


Activation of epinephrine-sensitive adenylate cyclase in rat liver by cytosolic protein-nucleotide complex.

The cytosolic fraction from rat liver enhanced the basal and glucagon-sensitive adenylate cyclase (EC 4.6.1.1) of hepatic plasma membranes and revealed its (R)-(-)-epinephrine sensitivity. Such phenomena were usually obtained by the addition of low concentrations of GTP to the medium employed for the cyclase assay. Comparative studies of the behavior of the cytosolic factor and GTP in response to various treatments were performed. We present evidence that the stimulatory activity of the soluble factor was reduced after treatment by alkaline phosphatase, by the nucleotide phosphohydrolases present in the plasma membranes, and by trypsin. These results strongly suggest that the soluble activator is a nucleotide-protein complex and further demonstrate that GTP may be of physiological significance in the regulation of the adenylate cyclase system.


Effects of free fatty acids on activity of hepatic microsomal 3-hydroxy-3-methylglutaryl coenzyme A reductase and on secretion of triglyceride and cholesterol by liver.

The output of triglyceride and cholesterol, and the activity of microsomal 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMG-CoA reductase) were measured following perfusion of the isolated rat liver with a medium containing free fatty acids. The activity of the enzyme and the output of cholesterol and triglyceride are regulated by the quantity and structure of the free fatty acid. The activity of HMG-CoA reductase was linearly proportional to uptake of oleic acid (18:1) by the liver. Output of triglyceride and cholesterol in the presence of oleate was also stimulated although, under these experimental conditions, the maximal secretory rate for cholesterol was observed while reductase activity was still increasing. When equimolar quantities of palmitic (16:0), oleic (18:1), or linoleic acid (18:2) were infused, the observed activity of the enzyme was in the order 18:1 greater than 18:2 = 16:0. Output of triglyceride was in the order 18:1 = 18:2 greater than 16:0. Output of free and esterified cholesterol was in the order 18:1 greater than 18:2 greater than 16:0. The stimulation by free fatty acids of hepatic cholesterogenesis, as estimated by the activity of HMG-CoA reductase, probably is the result of stimulation by free fatty acids of secretion of triglyceride in the very low density lipoprotein, and the obligatory requirement for cholesterol as a component of the lipoprotein.


Comparative properties of vertebrate parvalbumins.

Pure parvalbumins isolated from turtle, chicken, and rabbit white skeletal muscle have been characterized in terms of their physical, chemical, and immunological properties. As for the parvalbumins of most fish and amphibians, they have sedimentation constants S20,w of approximately 1.45 +/- 0.25 S and molecular weights of approximately 12,000, with little or no evidence for aggregation. They contain no tryptophan, at most one tyrosine, and a high proportion of phenylalanine, resulting in characteristic absorption spectra. All three parvalbumins contain 2 g atoms of calcium/mol bound with a KDiss less than or equal to 10(-6) M. Complete removal of calcium can be achieved by treatment with EDTA and EGTA or by a purified preparation of fragmented sarcoplasmic reticulum. By a direct analytical procedure, the concentration of parvalbumins in white skeletal muscle from the turtle, chicken, and rabbit was estimated at approximately 9 to 11, 0.2 to 0.4, and 0.6 to 1.1 g/kg, respectively. No parvalbumin or immunologically cross-reacting material could be detected in chicken white breast muscle, and very little was found in rabbit red muscle. All three proteins are immunologically distinct. A "minor" isoparvalbumin (approximately 2% of the major component) was found in turtle muscle only.


Calorimetric study of microsomal membrane.

Differential scanning calorimetric measurements of normal rat liver microsomes reveal a single thermal transition at about 50 degrees. This transition is assigned to irreversible prorein denaturation. There is no evidence for a reversible lipid phase transition at any temperature above 0 degrees, indicating that the microsomal membrane is in teh fluid state under these conditions. Rats fed a fat-free diet which increases the degree of saturation of fatty acids in the membrane lipids do produce microsomal membranes exhibiting a reversible lipid phase transition. The NADH=dependent and NADPH-dependent enzymatic reductions of cytochrome c show linear Arrhenius behavior in the normal rat liver microsomes but reveal discontinuities and breaks in the Arrhenius plots at approximately the calorimetrically determined phase transition temperatures in microsomes from rats fed the fat-free diet. Hence, the fluidity of cell membranes can be altered by diet with consequent effects on membrane-supported functions. The data further show that the lipid organization of the membrane is not independent of the protein component and supports models of membrane structure where a separate class of lipids forms a boundary between the bulk phase and the proteins.


Recombinations of subunits of Phaseolus vulgaris isolectins.

Phaseolus vulgaris phytohemagglutinin is formed in vivo by the combination of erythrocyte (E)-reactive and lymphocyte (L)-reactive subunits into five tetrameric isolectins:L4,L3E1, L2E2, L1E3, and E4. Evidence for phytohemagglutinin subunit structure is obtained by in vitro dissociation of native isolectins in 6 M guanidine HCl followed by removal of dissociating agents to allow subunit recombination. Dissociation and recombination of L4 yielded a single protein, electrophoretically indistinguishable from the native L4. Similar treatment of E4 also yielded a single protein indistinguishable from native E4. Treatment of L3E1, L2E2, L1E3, or a mixture of L4 and E4, yielded five distinct proteins electrophoretically similar to all five native phytohemagglutinin isolectins. Milligram quantities of all five recombinant isolectins were prepared either from L2E2 or a mixture of L4 and L1E3 proportioned to yield equimolar quantitives of the two subunits on dissociation. The recombinant isolectins were purified by affinity and SP-Sephadex ion exchange chromatography. Electrophoretic and chromatographic properties and the erythroagglutinating and mitogenic activities of recombinant isolectins were essentially identical with the native isolectins. The inclusion of 125I-labeled L4 in the dissociation results in a distribution of 125I-labeled L subunit among the purified recombinant isolectins proportional to their proposed subunit structures.


Steroid hormone receptors in normal human lymphocytes. Induction of glucocorticoid receptor activity by phytohemagglutinin stimulation.

The presence of specific steroid hormone receptors in human lymphocytes was investigated in unstimulated and phytohemagglutinin-stimulated glass wool column-purified peripheral blood lymphocytes. Specific steroid binding in intact cells was determined by a whole cell competitive binding assay. Non-phytohemagglutinin-stimulated lymphocytes had about 2700 specific glucocorticoid binding sites per cell; phytohemagglutinin stimulation induced a 2 to 3-fold increase in glucocorticoid receptor activity within 16 h of culture. No estrogen, androgen, or progestin binding sites were detected in either unstimulated or phytohemagglutinin-stimulated peripheral blood lymphocytes. Scatchard analysis of glucocorticoid binding was consistent with a single class of receptor sites with a dissociation constant (Kd) of about 5.5 x 10(-9) M (correlation coefficient r = -0.96). Significant competition for radiolabeled dexamethasone binding was not observed with steroids lacking glucocorticoid activity. There was good agreement between relative binding affinities of various steroids to glucocorticoid receptor in lymphocytes and ability of these steroids to inhibit phytohemagglutinin-stimulated thymidine incorporation.


Biosynthesis of mitochondrial DNA. Is 8 S DNA an artifact?

Sucrose density gradient fractionation of isolated rat liver mitochondrial DNA ordinarily yields two peaks, one at 39 S, the other at 27 S. However, when these mitochondria are first incubated with a labeled DNA precursor, a labeled peak at about 8 S is also observed. Is this low molecular weight 8 S DNA merely an artifact of contamination or breakdown, or is it a functioning part of the mitochondrial genome? That it is not a nuclear contaminant is shown by: (a) the absence of nuclei or nuclear fragments in active mitochondrial preparations; (b) the insensitivity of 8 S DNA synthesis to treatment of mitochondria with DNase and RNase; (c) the ability of inner membrane preparations to synthesize this DNA; (d) the ability of atractyloside to inhibit incorporation of [3H]dATP into 8 S and 39 S or 27 S DNA equally; (e) the labeling of 8 S DNA (as well as 39 S and 27 S DNA) but not of nuclear DNA after the administration in vivo of [3H]thymidine. The evidence that 8 S DNA is not an artifact resulting from DNA breakdown during mitochondrial incubation or DNA isolation is as follows: (a) 8 S DNA can be isolated from unincubated mitochondrial; (b) 8 S DNA becomes labeled when labeled DNA precursors are administered in vivo; (c) 8 S DNA biosynthesis continues in the complete absence of labeled 39 S or 27 S DNA (whose synthesis is repressed by ethidium bromide), making it unlikely that 8 S DNA is formed from the breakdown of 39 S or 27 S DNA; (d) substitution of milder methods of DNA extraction does not decrease 8 S DNA labeling; moreover, the usual conditions of extraction, when applied to purified 39 S and 27 S DNA, do not generate 8 S DNA, nor does an additional mitochondrial washing cycle; (e) the specific radioactivity of 8 S DNA is higher than that of 39 S or 27 S DNA, making it improbable that the latter forms are precursors of 8 S DNA. Since 8 S DNA is double-stranded, it is not identical to the 7 S fragment of D loop DNA. The hypothesis that the artifactual nicking of those DNA molecules which contain opposing D loops leads to the release of double-stranded fragments was tested. The DNA which was released was predominantly (and probably completely) single-stranded. We conclude that 8 S DNA is probably not an artifact and studies are in progress on its function.


Nonenzymatic glycosylation of hemoglobin.

The incubation of dialyzed hemoglobin A with a number of phosphorylated glycolytic intermediates leads to the formation of covalent hemoglobin adducts that co-chromatograph with hemoglobin AIb. Phosphorylated hexoses (glucose-6-P, fructose-6-P, fructose-1,6-P2) and trioses (glyceraldelyde-3-P, dihydroxyacetone-P) containing a free aldehyde or ketone can glycosylate hemoglobin A nonenzymatically. From 7 to 12% of the hemoglobin can be modified after a 72-h incubation of an equimolar mixture of hemoglobin A and the phosphorylated intermediate. No significant formation of adduct was seen with a sugar alone (glucose, fructose) or glycolytic intermediate which had a blocked aldehyde (glucose-1-P, glucose-1,6-P2, UDP-glucose). The addition of an equimolar amount of 2,3-diphosphoglycerate reduced adduct formation. Evidently, the phosphate is needed to orient and stabilize the intermediate in the bisphosphoglycerate pocket of hemoglobin so that the addition reaction can proceed. All of the hemoglobin A adducts were indistinguishable form hemoglobin AIb by ion exchange chromatography and isoelectric focusing. The hemoglobin A-glucose-6-P adduct and hemoglobin AIb had a NaB3H4-reducible linkage in the beta chain. The concentration of hemoglobin AIb is elevated in patients with diabetes mellitus. This presumably reflects the increased concentrations of glycolytic intermediates (glucose-6-P, fructose-6-P, fructose-1,6-P2, dihydroxyacetone-P) which were found to be significantly elevated in the red cells of diabetic patients as compared with normal controls.


Pseudosymmetry in the structure of myohemerythrin.

The three-dimensional structure of the protein myohemerythrin from retractor muscles of the sipunculan worn Themiste zostericola has been explored for the existance of approximately symmetry operators that locally interrelate portions of the molecule. First, the electron denisty distribution at 5.5 A resolution was examined. A local 2-fold axis that transposes the C-D helix pair into the A-B helix pair was found and refined by the method of least squares. The match in electron densities for a pure 2-fold rotation had a correlation coefficient of 0.56. Next, a comprehensive search was made for rotational symmetry in the Patterson function of an isolated molecule. The rotation function based on data to 6 A spacings showed a major peak, 72% of the self-peak height, that confirmed the result from electron density correlations. In addition, a pattern of lower level peaks revealed approximate point group symmetry as high as D4. Finally, the amino acid sequence has been inspected for evidence of a repeated structure. The level of amino acid identities between positions in the A-B and C-D helix pairs is 28%. Several factors are discussed to suggest that this homology, although low, is nonetheless significant.


Preparation and characterization of subfractions of bovine thyroid plasma membranes.

Two subfractions of bovine thyroid plasma membranes, light membranes (L-membranes) and heavy membranes (H-membranes), were obtained by a discontinuous sucrose gradient centrifugation of plasma membranes. Electron microscopy of the plasma membrane and its subfractions showed that the H-membranes were very similar to the plasma membrane fraction, both contained junctional complexes, long membrane sheets, and vesicles. In contrast, the L-membranes consisted mainly of short membrane sheets and vesicles, and only a few junctional complexes. The H-membranes had greater adenylate cyclase activity which responded to thyroid-stimulating hormone (TSH) while this hormone had very little effect on the enzyme activity in the L-membranes. Despite the marked difference in TSH stimulation of adenylate cyclase activity in the H- and L-membrane fractions, specific binding of 125I-TSH was similar in both fractions. The L-membranes had higher specific activities of 5'-nucleotidase and Mg2+ATPase while (Na+ + K+)-ATPase and alkaline phosphatase activities were similar in the two subfractions. Protein kinase activity of H-membranes was not significantly stimulated by exogenous cyclic adenosine 3':5'-monophosphate (cAMP). Plasma membranes and H-membranes contained a substrate capable of being phosphorylated. Such phosphorylation was slightly increased by addition of soluble protein kinase. The phosphorylation of exogenous histone by protein kinase of plasma membranes and H-membranes was augmented by cAMP. In contrast, L-membranes had very little protein kinase activity even when exogenous histone was added. They were not a very good substrate for cytosolic protein kinase.


Purification and characterization of core-associated polynucleotide 5'-triphosphatase from Vaccinia virus.

A core-associated enzyme, designated as polynucleotide 5'-triphosphatase, has been purified from vaccinia. Fractionation on ADP-agarose of the soluble extract from detergent-disrupted cores followed by chromatography on poly(U)-agarose produced an 80-fold purification of the enzyme. The enzyme has an approximate molecular weight of 113,000 and is composed of two polypeptides with approximate molecular weights of 90,000 and 26,000. Divalent metal ions are necessary for enzymatic activity, which occurs optimally at pH 8.4. The enzyme acts upon 5'-ATP- and 5'-GTP-terminated RNA and also on 5'-ATP-terminated poly(A), catalyzing the hydrolysis of only the gamma-phosphate. The presumed biological role of the enzyme based upon this specificity is the participation in the initial step in the sequence of reactions through which the primary 5' termini of vaccinia messenger RNA are capped with the groups m7G(5')ppp(5')NmpN-.


Immunological cross-reaction between calf and Drosophila histones.

Antibodies elicited by purified calf thymus histone fractions are used to quantitate the immunological cross-reactions between calf and Drosophila histones. The index of dissimilarity between calf and Drosophila histones are: H4, 1.0; H2B, 1.0; H3, 1.6; H2A, 2.0; H1, 16. These values respectively correspond to 0, 0, 4, 6, and 27% difference in amino acid composition. The differences in amino acid composition assessed by immunological techniques are in agreement with values obtained by amino acid analysis (Alfageme, C. R., Zweidler, A., Mahowald, A., and Cohen, L. H. (1974) J Biol. Chem. 249, 3729-3736 and Oliver, D., and Chalkley, R. (1972) Exp. Cell. Res. 73, 295-302). The strong immunological cross-reaction between calf and Drosophila histones H4, H3, H2A, and H2B leads us to suggest that antibodies elicited against purified histone fractions derived from calf thymus can serve as "universal" reagents for various studies of chromatin-bound histones. The use of standard preparations of antisera will facilitate comparisons of results obtained in different laboratories with various experimental systems.


A new antithrombogenic heparinized polymer.

A new antithrombogenic polymer was synthesized by photoinduced graft copolymerization of both a hydrophilic polymer and a cationic polymer to hydrophobic poly (vinyl chloride) copolymer, and by quarternizing and heparinizing the obtained graft copolymer. For polymers of various compositions obtained by the method described above, chemical composition, water absorption, membrane potential, and quantity of adsorbed heparin were determined and antithrombogenicity evaluated in vivo. Thus, it has been found that the polymer of excellent antithrombogenicity has a negative membrane potential, a moderate elution rate of heparin, adsorbs heparin in a quantity of approximately 15 wt-%, and has a degree of water adsorption of about 30 wt-%. Also, measurement of membrane potential was very useful for the estimation of the quantity of the adsorbed heparin and its change with time.


Effect of collagen crosslinking on the rate of resorption of implanted collagen tubing in rabbits.

Collagen tubes were tanned with glutaraldehyde for different periods of time. Some were oxidized with periodate and sterilized with either 60Co (1.5 Mrad) or propylene oxide. The tubes were coated with polyfilamentous polyester fabric, filled with x-ray contrast material, and implanted subcutaneously in rats and rabbits. Rate of resorption was ascertained by x-ray procedure of progressive leakage of contrast material. A close relation between tanning time of the collagen fabric-combined prostheses and rate of their resorption in subcutis was found in both rats and rabbits. In rabbits, however, the implants were resorbed at a significantly faster rate than in rats. No effect of oxidation of collagen on the resorption was observed. Collagen tubes tanned for shorter time periods and sterilized with propylene oxide were more resistant to degradation than those sterilized with irradiation. This difference was absent, however, with material tanned for longer times. The mechanism of resorption of implanted collagen tubes was studied by morphological methods. The role of inflammatory cells in resorption is documented. The paper indicates the advantages as well as limits of the x-ray method of studying the resorption rate of biodegradable materials.


Polytetrafluoroethylene-N-vinylpyrrolidone graft copolymers: affinity with plasma proteins.

N-vinylpyrrolidone was grafted onto films, grains, and tubes of polytetrafluoroethylene (PTFE) using a radiation procedure. By controlling the experimental conditions, it was possible to obtain an analogous grafting percentage on films and grains. Kinetic grafting onto a tube might be used under certain conditions to preserve both size and water tightness. Poly(vinylpyrrolidone) (PVP)-grafted material became hydrophilic and slightly swollen according to the grafting percentage obtained. A scanning electron microscope (SEM) analysis of polymer surfaces showed preferential polymerization sites at a low radiation dose. A regular, velvet-like surface was obtained for a high grafting percentage induced at a low radiation intensity. Biological properties of PTFE were compared before and after PVP grafting. PTFE appeared to be a nonreactive polymer with respect to the blood-clotting proteins. No significant change was observed using different granulometric sizes of PTFE grains, and its noteworthy inertness was preserved after PVP grafting. Interactions between polymer surfaces and human plasma proteins were analyzed using 125I-isolated proteins. Fibrinogen and immunoglobulins have a high affinity for PTFE; on the other hand, albumin was weakly adsorbed on the polymer surface. Marked changes in protein adsorption were observed after PVP grafting. Hydrophilic group grafting induced an increase of albumin adsorption and a decrease of fibrinogen adsorption. Prothrombin affinity was not significatly affected by modified PTFE. Studies of serum protein adsorption using solid-phase radioimmunoassays were developed with monospecific and polyvalent antisera. After incubation of the whole serum protein and polymer, immunoglobulins were found to exhibit the major part of bound protein radioactivity on the polymer surface. No evidence of increasing adsorption of immunoglobulins from rabbit serum after immunization by PTFE or PTFE/PVP was observed. The immunoglobulin adsorption was submitted to a competitive effect with the other serum proteins. It was possible to postulate the competitive action of albumin, particularly after biological property changes induced by chemical grafting.


Influence of the heat treatment of commercial amalgam alloys on the phase composition of the alloys and the creep of the resulting amalgams.

The influence of heat treatment of dental alloys at several temperatures on the phases present in the alloy and the creep of the resulting amalgam as well as the correlation between phase changes and the creep was investigated. A dependence of the creep on the temperature of the heat treatment of the alloy was established. Substantial phase changes were observed after the different heat treatments. No correlation was detected between the creep rate and the changes in phases present in the dental alloys. A thermodynamically oriented explanation of the relation between the creep of the amalgam and the state of (non-) equilibrium of the corresponding amalgam alloy was proposed and discussed.


Inhibition and enhancement of platelet aggregation by collagen derivatives.

Rat tail tendon tropocollagen rapidly caused the aggregation of blood platelets coincident with collagen multimerization. Several long-chain alkyl derivatives of collagen, however, were completely unable to induce platelet aggregation even at unusually high concentrations. These collagen derivatives varied in their polymerization properties , and the polymerization of tropocollagen derivatives was shown not necessarily to be a prerequisite for platelet aggregation. These collagen derivatives may enhance the antithrombogenic properties of collagenbased prosthese. A short-chain derivative, collagen-glycine ethyl ester, profoundly enhanced platelet aggregation in the absence of any measurable polymerization. This derivative may be useful as a growth-promoting burn dressing.


Porous acrylic cement.

The use of acrylic bone cement has a number of shortcomings, viz., high curing temperatures that can cause thermal necrosis, release of toxic monomer, and a less than perfect cement-to-bone bond. However, by modifying the cement composition through the addition of a soluble, nontoxic filler such as sucrose or tricalcium phosphate which does not impair the workability of the material during surgery, a significant improvement in the performance of the cement can be achieved. Because the filler replaces part of the acrylic components, less heat is generated during curing while the filler itself acts as a heat sink. Also, less monomer, proportional to the amount replaced by the filler, diffuses from the implant site. Upon elution of the filler, a porous cement will be obtained provided that a critical minimum percentage loading is exceeded so that the filler crystals will make physical contact with each other. The value of this percentage depends on both crystal modification and size. In the 125-175 micron sucrose crystal size range, the critical minimum percentage lies in the range of 20-28 wt% loading. Above 30%, the interconnecting pore size increases sharply to a value which allows good tissue ingrowth into the pores. The introduction of filler and pores causes a drop in strength, but the diametral tensile strength of modified cement containing up to 40% pores and sucrose lies between .7 and 1.5 kg/mm2, respectively, which is still in the same range as that of bone.


Biodegradability and tissue reaction of random copolymers of L-leucine, L-aspartic acid, and L-aspartic acid esters.

A series of copoly(alpha-amino acids) with varying percentages of hydrophilic (L-aspartic acid) and hydrophobic monomers (L-leucine, beta-methyl-L-aspartate, and beta-benzyl-L-aspartate) were implanted subcutaneously in rats and the macroscopic degradation behavior was studied. Three groups of materials (A,B,C) with different ranges of hydrophilicity were distinguished: A) hydrophobic materials showed no degradation after 12 weeks; B) more hydrophilic materials revealed a gradual reduction in size of the samples, but were still present after 12 weeks; and C) hydrophilic copolymers disappeared within 24 hr. The tissue reactions caused by the materials of group A resembled that of silicone rubber, whereas those of group B showed a more cellular reaction.


The strength of LTI carbon dental implants.

In vitro mechanical tests have been performed on a variety of LTI pyrolytic carbon blade-type dental implants, and the test results have been analyzed using an analytical model. Tensile stresses at fracture were calculated to be about 5 X 10(4) psi and 8 X 10(3) psi in the LTI carbon coating and the graphite substrate, respectively. These values are close to their respective fracture strengths. The fracture loads predicted by the model are in good agreement with data obtained from the mechanical tests and are higher than forces expected in mastication.


Internalization of lectins in neuronal GERL.

Conjugates of ricin agglutinin and phytohemagglutinin with horseradish peroxidase (HRP) were used for a cytochemical study of internalization of their plasma membrane "receptors" in cultured isolated mouse dorsal root ganglion neurons. Labeling of cells with lectin-HRP was done at 4 degrees C, and internalization was performed at 37 degrees C in a culture medium free of lectin-HRP. 15-20 min after incubation at 37 degrees C, lectin-HRP receptor complexes were seen in vesicles or tubules located near the plasma membrane. After 1-3 h at 37 degrees C, lectin-HRP-receptor complexes accumulated in vesicles and tubules corresponding to acid phosphatase-rich vesicles and tubules (GERL) at the trans aspect of the Golgi apparatus. A few coated vesicles and probably some dense bodies contained HRP after 3-6 h of incubation at 37 degrees C. Soluble HRP was not endocytosed under the conditions of this experiment or when it was present in the incubation medium at 37 degrees C. Internalization of lectin-HRP-receptor conjugates was decreased or inhibited by mitochondrial respiration inhibitors but not by cytochalasin B or colchicine. These studies indicate that lectin-labeled plasma membrane moieties of neurons are endocytosed primarily in elements of GERL.


Evaluation of indirect blood pressure measurement as a method of assessment of peripheral vascular disease.

In over 200 patients with peripheral vascular disease, pressure index (formula: see text) was compared with radiological grade; although calf P.I. had a better correlation, there was wide overlap between the groups. In 28 patients, resting calf P.I. was compared with functional impairment measured on a treadmill. At two rates of exercise, calf P.I. correlated poorly with functional impairment. In 35 patients, reproducibility of indirect pressure measurement was assessed; coefficients of variation ranged 0-10%; coefficient of variation of calf P.I. ranged 0-8%; coefficient of variation of calf P.I. ranged 0-8%. In 12 patients, indirect thigh systolic pressure was compared with and nearly always exceeded direct common femoral artery pressure; this was due to an effect of the cuff which reduced blood flow and reduced the pressure gradient. This methodological error has not previously been described and reduces the clinical value of the measurement.


An inflatable occluder for clinical use.

An arterial balloon occluder has been developed for use in surgical practice. Clinical and experimental assessment have confirmed it functions satisfactorily and is less traumatic than conventional clamps. It is less likely to crack atherosclerotic plaques as it occludes in the line of least resistance rather than crushing the artery from side to side. It also has other advantages. It gives precision control of arterial flow rather than the "on" or "off" effect of conventional clamps. The control is remote from the operating field and there are no clamp handles in the wound to catch on sutures. Occlusion around a catheter or cannula is easily achieved. Examples of its use in clinical practice are given which illustrate these points. We have found this to be a valuable additional vascular instrument.


Characterization of mitochondrial and cytoplasmic ribosomes from Paramecium aurelia.

The ribosomes extracted from the mitochondria of the ciliate, Paramecium aurelia, have been shown to sediment at 80S in sucrose gradients. The cytoplasmic ribosomes also sediment at 80S but can be distinguished from their mitochondrial counterparts by a number of criteria. Lowering of the Mg++ concentration, addition of EDTA, or high KCl concentrations results in the dissociation of the cytoplasmic ribosomes into 60S and 40S subunits, whereas the mitochondrial ribosomes dissociate into a single sedimentation class at 55S. Furthermore, the relative sensitivity of the two types of ribosome to dissociating conditions can be distinguished. Electron microscopy of negatively stained 80S particles from both sources has also shown that the two types can be differentiated. The cytoplasmic particles show dimensions of 270 X 220 A whereas the mitochondrial particles are larger (330 X 240 A). In addition, there are several distinctive morphological features. The incorporation of [14C]leucine into nascent polypeptides associated with both mitochondrial and cytoplasmic ribosomes has been shown: the incorporation into cytoplasmic 80S particles is resistant to erythromycin and chloramphenicol but sensitive to cycloheximide, whereas incorporation into the mitochondrial particles is sensitive to erythromycin and chloramphenicol but resistant to cycloheximide.


Large-sized polysomes in Chironomus tentans salivary glands and their relation to Balbiani ring 75S RNA.

Polysomes from the salivary glands of Chironomus tentans were investigated to determine whether Balbiani ring 75S RNA is incorporated into polysomal structures, and thus probably acts as messenger RNA. A new extraction technique for obtaining ribonucleoproteins was applied that gives a high yield of polysomes with only moderate degradation of the cytoplasmic, high molecular weight RNA. The polysomes sedimented in a broad region (200-2,000S) with a peak value of about 700S, which suggested that they were partly of very large sizes. This was confirmed by visualization of the polysomes in the electron microscope: 400S polysomes contained mainly 11-16 ribosomes, and 1,500S polysomes about 60 ribosomes per polysome. However, polysomes containing 100 or more ribosomes were also observed. It was further established that most of the cytoplasmic 75S RNA was located in polysomes, preferentially in the most rapidly sedimenting ones. From the available information on Balbiani ring RNA in cytoplasm and the present demonstration of 75S RNA molecules in polysomes, it was concluded that at least some Balbiani ring RNA, generated as 75S RNA within the Balbiani rings, eventually enters polysomes without being measurably changed in size. The present information on the potential amino acid coding sequences in 75S RNA is discussed in relation to the large size of the polysomes observed.


A surgical treatment with an "on-lay" patch for experimental myocardial infarction.

The authors proposed a new operative method for acute myocardial infarction with a prosthetic "on-lay" patch in a dog model, and functional and morphological studies were made in the acute and long survival dogs. In this method, the following advantages were observed: Improvement of cardiac functions, reduction of mortality, prevention of ventricular aneurysm, prevention of the occurrence of arrhythmias and ventricular fibrillation, minimum reduction of the intracardiac capacity and no suture dehiscence resulting in cardiac rupture. It is shown that this surgical method can be applied securely to the emergent cases of acute myocardial infarction failing to respond to medical therapy.


Membrane fusion during secretion. A hypothesis based on electron microscope observation of Phytophthora Palmivora zoospores during encystment.

Interpretation of freeze-fracture and thin-section results shows that fusion of the peripheral vesicle with the plasmalemma of a Phytophthora palmivora zoospore occurs at several discrete sites and results in the formation and expansion of a particle-free bilayer membrane diaphragm and in the appearance of a polymorphic network of membrane-bounded tunnels, the lumina of which are continuous with the cytoplasm. The outer half of the bilayer membrane diaphragm appears continuous with the outer half of the plasma membrane; the inner half of the bilayer membrane diaphragm with the inner half of the peripheral vesicle membrane; and the inner half of the plasmalemma with the outer half of the peripheral vesicle membrane. Interpretation of our results leads us to formulate a hypothesis for a sequence of several intermediate stages involved in membrane fusion. The initial fusion event is viewed as a local catastrophe (Thom, R. 1972. Stabilité Structurelle et Morphogenèse. W. A. Benjamin Inc., Reading, Mass.) involving the sudden reorganization of apposed elements of the inner half of the plasmalemma and the outer half of the peripheral vesicle membrane. Fusion of apposed components at the rim of the perimeter of fusion results in the formation of a toroid hemi-micelle which provides continuity between the inner half of the plasmalemma and the outer half of the peripheral vesicle membrane. Simultaneously, apposed components at the site of fusion may reorganize into an inverted membrane micelle. A bilayer membrane diaphragm is then formed by apposition and flowing of components form the outer half of the plasmalemma and the inner (exoplasmic) half of the peripheral vesicle membrane. The existence of large areas of membrane contact before fusion may lead to several fusion events and the formation of a polymorphic network of membrane-bound tunnels.


Concentration of elements in mitotic chromatin as measured by x-ray microanalysis.

Unfixed frozen-dried and uncoated tissue sections of the mouse duodenum were placed on carbon planchets and analyzed in a scanning electron microscope fitted with energy dispersive X-ray equipment. Computer analysis of the X-ray spectra allowed elemental microanalysis of the nucleus, cytoplasm, and mitotic chromatin regions in the cryptal and villus enterocytes. The peak to continuum ratio of S, Cl, K, and Ca were higher in mitotic chromatin than any of the other sites measured. The redistribution of Ca at mitosis is postulated to help explain both chromosome condensation and assembly of the mitotic spindle apparatus.


Raynaud's phenomenon following long-term repeated action of great differences of temperature.

The aim of these investigations was to study the effects of chronic thermal trauma on the development of vasomotor disturbances of the hands. The investigations were performed on 597 workers of the Fishing Company. The incidence of vasomotor disturbances of the hands was compared in: (1) workers not exposed to thermal trauma; (2) workers with long-term exposure to cold; (3) workers exposed to alternating influence of cold and heat. Superficial temperature was determined, finger plethysmography, capillaroscopy and hand arteriography were carried out. The investigations demonstrated that long-term alternating exposure to thermal trauma causes development of vasomotor disturbances. Clinical manifestations of Raynaud's syndrome were found in nearly 50% of female workers in fish processing plant whose hands were exposed to the action of ice and hot water. The incidence of vasomotor disturbances in workers exposed to long-term effects of cold was low.


Biochemical characterization of RNA and protein synthesis in erythrocyte development.

Newts (Triturus cristatus) made anemic with acetylphenylhydrazine (APH) fail to regenerate erythrocytes (RBC's) immediately and exhibit a latent period of 1.5-2 wk during which animals lack RBC's and are aplastic. With the establishment of erythroid regeneration at 10-14 days, relatively homogeneous populations of successive erythropoietic stages occur in the blood. This feature makes possible biochemical analyses of events in early, intermediate, and late developmental stages, respectively, each of which can be obtained in vivo with minimal contamination by other stages. Previous studies have described a primitive cell population referred to as "erythroid precursor cells" (EPC's) which precedes the appearance of definitive erythroid elements. The present studies show that EPC's and early erythroid cells are engaged mainly in ribosomal production, including synthesis of rRNA and ribosomal proteins. Moreover, EPC's and early erythroid cells also synthesize tRNA and a presumed Hb-mRNA which has been identified by its sedimentation rate at 9-12 s and its content of polyadenylic acid. In intermediate stages, there occurs a fourfold decrease in the level of RNA synthesis and, while rRNA continues to be formed, there is a disproportionate accumulation of the two major cytoplasmic rRNA species in favor of the large ribosomal subunit RNA. In late developmental stages, the level of RNA synthesis is markedly diminished with little or no evidence of formation of defined RNA classes. Correlated radioautographic and biochemical studies with radioactive delta-aminolevulinic acid and leucine indicate that EPC's and other early erythroid elements synthesize not only hemoglobin but also ferritin and ribosomal proteins. It is concluded that: (a) erythroid RNA synthesis is most pronounced in the early developmental stages, being manifested predominantly by rRNA production but including tRNA and Hb-mRNA; (b) intermediate developmental stages show both "ribosomal wastage" and decreased growth rate, marking a pivotal point between the transcriptional activities of early stages and translational activities of late stages; (c) EPC's represent a cell population already committed to RBC formation and are excluded from a role as the pluripotential stem cell.


Redox constituents in milk fat globule membranes and rough endoplasmic reticulum from lactating mammary gland.

Milk fat globule membranes (MFGM) and rough endoplasmic reticulum (RER) membranes were isolated from milk and lactating mammary gland from the cow and were characterized by biochemical and electron microscope methods in terms of gross composition (proteins, phospholipids, neutral lipids, cholesterol, RNA, and DNA) and purity. Both fractions contained significant amounts of a b-type cytochrome with several properties similar to those of cytochrome b5 from liver, as well as a rotenone-insensitive NADH- and NADPH-cytochrome c reductase. The b-type cytochrome content in the apical plasma membrane-derived MFGM was of the same order of magnitude as it was in RER membranes. It was characterized by a high resistance to extraction by low- and high-salt concentrations and nonionic detergents. MFGM contained much more flavin and much higher activities of xanthine oxidase than the RER membranes. The same redox components were found in MFGM and mammary RER from women, rats, mice, and goats, but in absolute contents great differences between the species were noted. The cytochromes described here differed from liver cytochrome b5 in some spectral properties. The alpha-band of the reduced hepatic cytochrome b5 is asymmetric with a maximum at 555 nm that is split into two distinct peaks at low temperatures. The alpha-band of the b-type cytochromes from MFGM and mammary RER appears as one symmetrical peak at about 560 nm that is not split at low temperatures. When treated with cyanide, MFGM and mammary microsomes showed difference spectra of a reduced b-type cytochrome. Under the same conditions, liver microsomes gave a completely different spectrum. These findings demonstrate the presence of a b-type cytochrome and associated redox enzymes in MFGM, i.e., a derivative of the apical cell surface membrane that is regularly used for envelopment of the milk fat globule during secretion.


Role of microtubule assembly in lysosomal enzyme secretion from human polymorphonuclear leukocytes. A reevaluation.

The dose-related inhibition by colchicine of both lysosomal enzyme release and microtubule assembly was studied in human polymorphonuclear leukocytes (PMN) exposed to the nonphagocytic stimulus, zymosan-treated serum (ZTS). Cells were pretreated with colchicine (60 min, 37 degrees C) with or without cytochalasin B (5 microng/ml, 10 min) and then stimulated with ZTS (10%). Microtubule numbers in both cytochalasin B-treated and untreated PMN were increased by stimulation and depressed below resting levels in a dose-response fashion by colchicine concentrations above 10(-7) M. These concentrations also inhibited enzyme release in a dose-response fashion although the inhibition of microtubule assembly was proportionately greater than the inhibition of enzyme release. Other aspects of PMN morphology were affected by colchicine. Cytochalasin B-treated PMN were rounded, and in thin sections the retracted plasma membrane appeared as invaginations oriented toward centrally located centrioles. Membrane invaginations were restricted to the cell periphery in cells treated with inhibitory concentrations of colchicine, and the centrioles and Golgi apparatus were displaced from their usual position. After stimulation and subsequent degranulation, the size and number of membrane invaginations greatly increased. They remained peripheral in cells pretreated with greater than 10(-7) M colchicine but were numerous in the pericentriolar region in cells treated with less than 10(-7) M. Similarly, untreated PMN that were permitted to phagocytose immune precipitates had many phagosomes adjacent to the centriole. After colchicine treatment, phagosomes were distributed randomly, without any preferential association with the centrioles. These data suggest that microtubules are involved in maintaining the internal organization of cells and the topologic relationships between organelles and the plasma membrane.


Disappearance of afferent and efferent nerve terminals in the inner ear of the chick embryo after chronic treatment with beta-bungarotoxin.

Beta-Bungarotoxin(beta-BT) was applied to chick embryos at 3-day intervals beginning on the 4th day of incubation to see the effect of chronically and massively applied beta-BT, and to investigate the hair cell-nerve relationship in the developing inner ear by electron microscopy. On the 10th day of incubation, nerve terminals had achieved contact with differentiating hair cells, but the acoustico-vestibular ganglion cells of treated animals were decreased in number to one-third of those of the control. By the 14th day, most of the ganglion cells degenerated and disappeared, and only a few nerve terminals were seen in the neuroepithelium. At this time, most of the hair cells lacked synaptic contacts with nerve terminals; but their presynaptic specialization remained intact and they showed evidence of continuing differentiation. On the 17th day, the acoustico-vestibular ganglion cells were completely absent. All the hair cells were devoid of afferent and efferent innervation but were fully differentiated on the 21st day. Beta-BT was found to have a similar destructive effect on cultured spinal ganglion cells. The present study shows that beta-BT kills acoustico-vestibular and spinal nerve cells when applied chronically and massively during development. Furthermore, the differentiation of hair cells proceeds normally, and their presynaptic specializations are maintained when nerve terminals are absent during later developmental stages.


Characterization of the cell cycle of cultured human diploid cells: effects of aging and hydrocortisone.

Age-related changes in the cytokinetics of human diploid cells in vitro have been compared in normal cultures and in cultures in which lifespan has been prolonged by the addition of hydrocortisone to the medium. For both cultures, with advancing age the fraction of cells in the actively proliferating pool decreased and the intercellular variation in cell cycle times increased. The average cell cycle time was prolonged during aging due almost entirely to changes in the duration of G1. The duration of S remained constant, while a small delay in G 2 was observed in late passage cells near the end of their lifespan. Although the same pattern of change in proliferative parameters occurred in both control and hydrocortisone-treated cultures, the changes were somewhat delayed in the presence of the steroid. The results are interpreted in terms of several cell cycle models and suggest that the events controlling cell proliferation are sensitive to hydrocortisone modulation during the G1 and possibly the G2 periods.


Studies on a fractionated murine fibrosarcoma: a reproducible method for the cautious and a caution for the unwary.

A technique is described for the dissociation and fractionation by isopycnic centrifugation (4,000 x g, 60 minutes, 4 degrees C) in linear bovine albumin density gradients (12 ml, pH 5.2 real osmolality 333 mmol/Kg water, 1.030-1.075 g/cm3) of cells ( less than or equal to 3 x 10(7)/gradient) released by a strictly standardised combination of mechanical and enzymatic means from a transplantable methylcholanthrene induced BALB/c fibrosarcoma. Optimal conditions for reproducible localisation of cell bands with maintenance of both satisfactory resolution and satisfactory viable cell recovery ( greater than 80%) were established by means of a series of simultaneous double fractionation experiments. When rebanding was performed under these conditions the density of the median of the cell count of the refractioned cells shifted less than 0.0005 g/cm3. Experiments also showed that close adherence to certain aspects of the tissue dissociation and fractionation protocol was necessary to avoid reduced cell yields and viabilities, density-dependent selective cell lossses, reductions in resolution and shifts in the location of cell bands. Other aspects of the protocol were tolerant to variation without introducting artefacts.


Characterization of a depolarizing dopamine response in a vertebrate neuronal somatic cell hybrid.

The physiology and pharmacology of a depolarizing dopamine response was studied in the vertebrate neuronal somatic cell hybrid TCX11. The average resting membrane potential was -50 mV (S.D.=+/-7) with a membrane resistance of 40.5 mOhms (S.D.=+/-8) as determined from intracellular recordings. Depolarizing current pulses did not elicit an action potential. Cells displayed a linear current-voltage relationship when artificially depolarized up to +30 mV. Iontophoretically applied dopamine elicited a depolarizing response with a conductance increase and a reversal potential of -15 mV (S.D.=+/-4.7). Experiments altering medium ion concentrations demonstrated the conductance increase was to sodium and most likely potassium. The dopamine agonist ET495 (Piribedil) and the analogue epinine mimicked dopamine, while closely related biogenic amines, with the exception of noradrenaline, elicited no response. Apomorphine also elicited a depolarizing response but was much less efficacious than Piribedil. Noradrenaline was less potent than dopamine and appeared to act at the dopamine receptor. Methylation (3-methoxytyramine) or absence of the 3-hydroxy group (tyramine) of dopamine resulted in total loss of activity. The dopamine antagonists chlorpromazine, trifluoperazine, promazine, and bulbocapnine reversibly blocked the response to dopamine at medium concentrations less than 5 micronM. The adrenergic antagonist phentolamine blocked the response while phenoxybenzamine only reduced the response at higher concentrations. The acetylcholine antagonists alpha-bungarotoxin, hexamethonium, and scopolamine did not block the dopamine response. Both d-tubocurarine and atropine acted as antagonists. Collectively, these results demonstrate the presence of a receptor on a cultured cell line that is specific for dopamine, mediates a depolarizing and conductance increase response to dopamine, and displays the pharmacology most closely associated with dopamine receptors.


Expression of liver and placental alkaline phosphatases in Chang liver cells.

This paper presents evidence that a protein characteristic of differentiated liver cells, liver alkaline phosphatase is synthesized by the Chang liver cell line. Liver alkaline phosphatase was demonstrated by immunochemical assay, 32P-labeling and polyacrylamide gel electrophoresis, immunofluorescence microscopy, and the fluorescence-activated cell sorter. The synthesis of the liver enzyme by the Chang liver cells is interpreted to indicate fidelity of the Chang cells to their origin from human liver tissue. Chang liver cells also synthesize a phosphatase which is similar if not indentical to the placental alkaline phosphatase. Since a placental-type alkaline phosphatase has been observed in a number of non-trophoblastic cell lines and also in some neoplasms, it does not seem reliable as an index of the origins of the cell line. Because of the claims that Chang liver cells are actually HeLa cells, HeLa cells were studied in tandem with the Chang cells. The results showed that the HeLa cells do not make the liver type phosphatase. The data are discussed in relation to the question of HeLa cell contamination of the Chang cell line and the validity of criteria normally used to identify cell lines.


Erythroid colony formation in cultures of human marrow: effect of leukocyte conditioned medium.

A method, based on the differing capacities of cells to adhere to a column of polyester fibres, has been described for separating human bone marrow cells into a nonadherent and an adherent fraction. The effect of this cell separation procedure on colony formation by erythroid progenitor cells was investigated. In contrast to the unseparated population, it was found that erythropoietin-dependent erythroid colony formation by nonadherent cells could be considerably enhanced by the addition of leukocyte conditioned medium to the cultures. Similar erythroid enhancing activity was also detected in a partially purified preparation of granulocytic colony stimulating activity obtained from human embryo kidney culture supernatants. Erythroid colony formation in the absence of added erythropoietin, by non-adherent bone marrow cells from patients with polycythemia rubra vera, were also enhanced by the addition of LCM to the cultures. This finding suggests that the enhancing factor in LCM may not be dependent on the presence of erythropoietin in the cultures for its activity. While the cellular mechanisms by which leukocyte conditioned medium enhances erythroid growth remain to be determined, the data presented provides strong evidence for the view that the plating efficiency of erythroid progenitor cells is determined not only be the concentration of erythropoietin, but also by the presence of leukocyte conditioned medium in the cultures.


The effect of 2-deoxyglucose on guinea pig polymorphonuclear leukocyte phagocytosis.

The effects of 2-deoxyglucose (DOG), an inhibitor of glycolysis, on guinea pig polymorphonuclear leukocytes (PMN) obtained from peritoneal exudates was examined. ATP levels in PMN were reduced by 40% by one hour following an incubation with 2-deoxyglucose. When complement (C3) coated 14C-staphylococcus aureus, C3 coated lipopolysaccharide-paraffin oil droplets (LPS-PO), 14C-pneumococcus opsonized with IgG, or albumin coated paraffin oil droplets opsonized with IgG were added to cell suspensions containing DOG, the phagocytizing rate was 1,310+/-55 cpm/5 X 10(6) cells/15 minutes, 6+/-2 microng paraffin oil (PO)/10(7) cells/minute, 2,250+/-175 cpm/1 X 10(6) cells/20 minutes or 0.037+/-0.01 mg PO/10(7) cells/minute compared to control values of 5,970+/-275 cpm/5 X 10(6) cells/15 minutes, 35+/-3 microng PO/10(7) cells/15 minutes, 4,510+/-200 cpm/1 X 10(6) cells/20 minutes and 0.067+/-0.01 mg PO/10(7) cells/minute. In parallel studies the phagocytic index for latex was 0.74+/-0.28 in DOG compared to control of 2.36+/-1.13 and the phagocytic rate of albumin coated paraffin oil droplets was 0.029+/-0.01 mg PO/10(7) PMN/minute in DOG compared to control of 0.048 mg PO/10(7) cells/minute. When ATP levels were maintained by the simultaneous addition of 5 mM glucose or pyruvate to media containing DOG, latex ingestion was improved to 1.15+/-0.3 with glucose and 1.59+/-0.64 with pyruvate and albumin coated particles to 0.045+/-0.01 mg PO/10(7) PMN/minute with pyruvate. There was no improvement in the uptake of either the C3 dependent particles or IgG coated Pneumococci in media containing DOG and glucose and/or pyruvate. Following the removal of DOG from the extracellular medium and the addition of pyruvate or glucose, phagocytosis of C3 dependent LPS-PO was restored to normal values. Neither the binding of C3 or IgG coated particles to the PMN nor the lateral movement of glycoprotein utilizing concanavalin A capping was affected by DOG. Thus, the presence of DOG in the PMN containing adequate amounts of ATP will selectively and reversibly inhibit those surface events required for phagocytosis of C3 and IgG bound particles but not latex particles or albumin particles which non-specifically bind to PMN.


The information content of titles in contraception literature.

This paper attempts to evaluate the effectiveness of a keyword or descriptor in the title, from the viewpoint of document selection and information retrieval for scientists engaged in contraception research. A total of 2152 document titles recorded in the Index Medicus during 1973-75 was examined. Another set of 567 contraception titles published in the Index Medicus during 1963-65 was also studied to determine if there were any changes in the information content of the titles over a 10-year period. Employing the Montgomery-Swanson method, it was found that 92.2% of the documents from the later period and 88.0% of those from the earlier period had at least one "contraception term" in their titles. The data obtained in this study exceed those of published reports. A brief review of some of the previous studies dealing with the retrieval capabilities of titles in various branches of medical and life sciences is presented. It is concluded that the title search method is a very effective means of retrieving pertinent documents in the field of contraceptive technology. A trend of improvement in the information content of the scientific titles is also noted.


Gas-liquid chromatography of nucleosides. Effect of silylating reagents and solvents.

The aims of this investigation were to study the completeness of silylation of nucleosides with three different reagents, bis(trimethylsilyl)trifluoroacetamide (BSTFA), bis(trimethylsilyl)acetamide (BSA) and trimethylsilylimidazole (TMSI), and to investigate the effect of different solvents (acetonitrile, pyridine, dimethylformamide, chloroform, methylene chloride, hexane, benzene, and toluene) on quantitation of derivatization. Closed-tube silylations of the nucleosides were performed with BSTFA, BSA, and TMSI, and for the most complete silylation, the optimal time, temperature, and molar excess of reagent were: for BSTFA, 150 degrees-15 min and 225 molar excess; for TMSI, 60 degrees-3 h and 1000 molar excess; and for BSA, 120 degrees-2 h and 250 molar excess. Also, silylations of seven major and minor nucleosides were carried out using a 1000 molar excess of BSTFA, BSA, and TMSI at 25 degrees with 5 min sonication, and at optimal silylation conditions as described above for the three reagents. The silylating strengths were determined by the increase in RWR (= weight response of nucleoside/weight response of pyrene) values, and are summarized for the amino group containing nucleosides silylated at room temperature as BSTFA greater than TMSI greater than BSA, and for silylation under optimal conditions as BSTFA greater than BSA greater than TMSI. The efficiency of silylation for the hydroxyl group-containing nucleosides silylated at room temperature was BSTFA greater than TMSI greater than BSA, and for silylation under optimal conditions BSTFA greater than TMSI = BSA...


A comparison of reversed-phase and partition high-performance liquid chromatography of some digitalis glycosides.

Comparison of the data for adsorption and reversed-phase chromatography of digitalis glycosides shows the complementary nature of the two modes of separation. The correct choice for a particular problem should make possible a rapid and good separation with simple isocratic systems. Detection limits vary between 10 and 100 ng per injection and permit the analysis of by-products even in low-dosage pharmaceutical formulations. Quantitation is easily possible with both chromatographic techniques using external standardization. The reproducibility for repetitive chromatograms is about 1% relative standard deviation for manual injections by loop injectors and is even significantly better for automatic injection. Reversed-phase chromatography can offer some advantages with regard to sample preparation of pharmaceutical formulations.


Separation of quinones and their derivatives by high-performance liquid chromatography.

The separation of mixtures of quinones by high-performance liquid chromatography has been studied. The technique described is suitable for separating differently substituted quinones. Particularly convenient is the conversion of quinones into 2,4-dinitrophenylhydrazones, which makes the reliable identification of benzoquinones and naphthoquinones possible. Studies of the separation of 2,4-dinitrophenylhydrazones of quinones and nitrophenols on Sephadex LH-20 gel indicated that the substances in question were not separated on the basis of differences in molecular weight; but the RF values were proportional to the dissociation constant.


Gas chromatographic separation of histamine and its metabolites.

A new, rapid and simple gas-liquid chromatographic method is described for the simultaneous determination of histamine, 1-methylhistamine, 4-imidazoleacetic acid and 1-methyl-4-imidazoleacetic acid. These compounds are derivatized at 60 degrees in one fast 30-min reaction with bis(trimethylsilyl)acetamide-trimethylchlorosilane and are then separated on OV-17. No previous methylation of the acids is needed. A complete profile of the four metabolites can be obtained in 8 min with baseline resolution, and there are no tailing effects. The quantitative response curves are linear in the nanogram range, with detection limits of the order of 10 ng for the acids and 100 ng for the amines. The structures of these new trimethylsilyl derivatives have been verified by combined gas-liquid chromatography-mass spectrometry.


Gas chromatographic determination of hydrocodone in serum.

A procedure for the determination of hydrocodone (dihydrocodeinone) in serum has been developed. Hydrocodone and N-isobutyldihydronorcodeinone, the internal standard, are extracted from serum by chloroform-isopropyl alcohol (9:1, v/v). The extracts are purified by back-extraction into 0.1 N sulfuric acid and a final basic extraction into benzene. The pentafluorophenylhydrazone derivatives are formed and determined using electron capture gas chromatography. As little as 1 ng/ml of hydrocodone in serum can be determined. A closely related compound and potential metabolite, dihydronorcodeinone, does not interfere. Serum hydrocodone levels were determined in dogs after oral and intravenous doses of 0.5 mg/kg, and in humans after a 10-mg oral dose of the bitartrate. A mean peak serum drug concentration of 23.6 ng/ml and a terminal half-life of 3.8 h resulted from the human study. The terminal half-life in serum was 1.8 h after the intravenous dose in dogs.


[Preparative approach to mannobiose and laminaribiose (author's transl)].

For synthetic purposes mannobiose is isolated from the polysaccharide mannan of ivory nut flower. The chain length of mannan A is determined to approximately 15 mannose units on average with a definite distribution from 5 to 25 mannose units. Acetolysis is optimized and yields 41% mannobiose. The separation on Sephadex G-15 yields pure mannobiose, and in addition its higher homologues in gram amounts. Detection of the eluates is performed using a differential refractometer. Correspondingly laminaribiose can be obtained from the polysaccharides laminaran (brown algae Laminaria hyperborea) and pachyman (fungus Poria cocos Wolf, Bukuryo) by selectively optimized acetolysis in 30% yield. Depending on the starting material separations are favourably performed on silica gel or on Sephadex G-15. Laminaribiose and its higher homologues are obtained in gram amounts.


The use of omicron-phthalaldehyde for fluorescence detection in conventional amino acid analyzers. Sub-nanomole sensitivity in the analysis of phenylthiohydantoin-amino acids.

A conventional amino acid analyzer has been modified for fluorescence detection using omicron-phthalaldehyde. The original ninhydrin system is retained, and the amino acid analyzer can be used with either of the detection systems. The fluorescence detection is used for identification of PTH-amino acids after back hydrolysis, when nanomole amounts of proteins are sequenced. Impurities in the water and the chemicals used for buffer preparations are limiting factors for the sensitivity of the fluorescence detection system. A 100-fold increase in sensitivity above the original ninhydrin system is obtained.


Profiles of prostaglandins A, B, E and F (series I and II) obtained by gas chromatography with multiple-ion detection.

A new method is presented for the simultaneous one-step derivatization of prostaglandins A1, A2, B1, B2, E1, E2, F1alpha and F2alpha with N,O-bis(trimethylsilyl)trifluoroacetamide-piperidine. The procedure provides stable and reproducible trimethylsilyl derivatives that show good chromatographic properties on OV-17 and OV-225 columns, and precludes any significant degree of interconversion of the prostaglandins. The prostaglandins F, B and E are determined as the corresponding tetrakis(trimethylsilyl), bis(trimethylsilyl) and 9-enol-tetrakis(trimethylsilyl) derivatives, respectively. The enolization of prostaglandins E proceeds quantitatively only if piperidine is added to the acetamide. This mixture also produces the novel derivative 9-piperidyl-tris(trimethylsilyl)prostaglandin A. Evidence is also presented on the quantitative injection losses due to the type of syringe used (1micronl or 10 micronl). A modified extraction procedure has been applied to samples of human seminal fluid. The procedure, which involves the centrifugation of the samples, allows the recovery of all of the prostaglandins in one fraction, which is then derivatized and screened by combined gas-liquid chromatography-multiple-ion detection techniques in order to identify the individual compounds.


Reversed-phase high-performance liquid chromatographic analytical system for aflatoxins in wines with fluorescence detection.

An improved procedure using reversed-phase high-performance liquid chromatography (HPLC) with fluorescence detection is described for the analysis of six aflatoxins (B1, B2, G1, G2, B2alpha, and G2alpha). The method provides greater analytical speed, reproducibility, and resolution, as well as better accuracy and sensitivity than the previously available thin-layer chromatographic methods. Aflatoxins B1 and G1 were hydrated to enhance their fluorescence response. Analyses of both hydrated and unhydrated compounds by HPLC provided simultaneous qualitative and quantitative information. A typical HPLC run was complete in 15 min. The recoveries of four aflatoxins (B1, B2, G1, and G2) from wines at the spiked level of 1 mug/l (1 ppb) were satisfactory. The method as applied to wine samples is sensitive at the 0.2 mug/l level.


Fatty acids. XIII. The gas-liquid chromatographic behaviour of dimethylene-interrupted methyl cis-octadecenynoates.

All of the dimethylene-interrupted methyl cis-octadecenynoate isomers, except the delta13c,17a isomer, have been prepared by partial hydrogenation of the corresponding methyl octadecadiynoate. Gas-liquid chromatographic analysis of the octadecenymoates has been carried out on Carbowax 20M, DEGA, DEGS, FFAP and Silar 10C stationary phases. The delta3,7 isomers decompose on chromatography. The equivalent chain lengths of all of the remaining methyl cis-octadecenynoates are reported.


Separation of gentamicin C-complex into five components by Craig distribution.

A gentamicin C-complex preparation was separated preparatively into five components by Craig distribution. Gentamicins C1, C2, and C1a were the major components, whereas components C2a and C2b represented only 4% of the total C-complex mixture. Paper chromatographic analysis showed that the gentamicin C2b separated by Craig distribution was identical with a gentamicin isolated from Micromonospora purpurea var. JI-33 fermentation broth and identified as 6'-N-methylgentamicin C1a. Similarly, component C2a was identical with a previously separated gentamicin component tentatively identified as the 6'-C epimer of gentamicin C2.


Isolation and determination of chlorinated organic pesticides by thin-layer chromatography and the application to toxicological analysis.

A sensitive method for the isolation and clean-up of chlorinated organic pesticides from tissues and biological materials is discussed. RF values of the pesticides in 26 solvent systems, and colours of the spots given with various chromogenic reagents, are reported. The limits of detection, and densitometric measurements, are also discussed. The average recovery has been found to be 94%. The distribution of endrin in different autopsy tissues from five different victims has been studied by the proposed technique.


New gas chromatogrphic assay for the quantification of methadone. Application in human and animal studies.

A new gas chromatographic assay utilizing 2-dimethylamino-4,4-diphenyl-5-nonanone as the internal standard was developed for the quantification of methadone. The method involved extraction of methadone with 1-chlorobutane from tissue at pH 9.8, re-extraction of an aliquot of the organic solvent with 0.5 M sulphuric acid, alkalinization and final extraction into chloroform. The assay was used to determine the concentration of methadone (i) in whole blood samples from a normal volunteer following a single 9.4-mg oral dose of d-methadone hydrochloride, (ii) in whole blood, saliva and gastric juice from a methadone addict maintained on 90 mg of dimethadone hydrochloride per day, (iii) in mouse liver microsomes incubated with methadone, and (iv) in the perfusate of the isolated perfused rat liver.


Simultaneous determination of anticonvulsant drugs by gas-liquid chromatography.

A quantitative gas-liquid chromatographic method for the determination of blood levels of ethosuximide, phenobarbitone, primidone and diphenylhydantoin is described. All four compounds are determined using 1 ml of serum. A simple, direct extraction technique is employed. Ethosuximide is analysed without derivatization. Subsequent flash alkylation with trimethylanilinium hydroxide allows the simultaneous determination of phenobarbitone, primidone, and diphenylhydantoin.


Automated high-resolution gas chromatographic analysis of psychotropic drugs in biological fluids using open-tubular glass capillary columns. I. Determination of nomifensine in human plasma.

An automated high-resolution gas chromatographic method has been developed for the determination of low levels of the antidepressant psychotropic drug Nomifensine in human plasma. The drug is extracted from alkalinized plasma with diethyl ether and then back-extracted into an acidic aqueous phase. After subsequent extraction into diethyl ether the drug is analysed by gas-liquid chromatography as its heptafluorobutyrate derivative using an OV-101 open-tubular glass capillary column with a nitrogen-specific detector. The propyl and butyl analogues of Nomifensine are used as internal standards, added to the plasma before extraction. The method is accurate, specific and precise, and capable of measuring plasma concentrations down to a level of 2 ng/ml. A preliminary study of the pharmacokinetics of the drug, together with steady-state level measurements in normal individuals receiving therapeutic dosages of Nomifensine, has been made.


Determination of doxorubicin hydrochloride in pharmaceutical preparations using high-pressure liquid chromatography.

An improved high-pressure liquid chromatographic method for the determination of doxorubicin hydrochloride (Adriamycin TM) in pharmaceutical preparations has been developed. A liquid-solid system using microporous silica (Zorbax) as the stationary phase and 3.8% 0.5 M sodium acetate buffer (pH 4.5) in isopropanol as the mobile phase gave optimum separation of doxorubicin hydrochloride from the excipient (lactose) and minor impurities. The doxorubicin hydrochloride content of a 10-mg vial can be determined in 25-30 min with a relative standard deviation of 3.2%. Results compare favorably with those obtained by established thin-layer chromatography and spectrophotometric procedures. The sensitivity of the method is sufficiently high (nanogram range) for possible use in metabolic or pharmacological studies.


Ligand-exchange separations of amino acids. I. Distribution equilibria of some amino acids between ammoniacal copper(II) nitrate solutions and phosphonic, carboxylic and iminodiacetic ion exchangers in the copper(II) form.

The ligand-exchange process for the distribution of glycine, alanine and leucine between a copper(II) phosphonate resin and ammoniacal solutions of copper nitrate has been studied. The formation in both resin and solution phases of mixed CuA(NH3)+i complexes (i = 1-3) was demonstrated. A theoretical expression for the distribution coefficient, taking into account the various parameters upon which it depends, has been established and verified experimentally. Some experiments carried out with carboxylic and iminodiacetic resin in the Cu(II) form confirmed the validity of the theoretical model. The distribution of an amino acid by ligand exchange occurs as a complex phenomenon that brings many equilibria into play. However, by simplifying the theory with some approximations, it is possible to describe the distribution by means of two equilibria, one of ion exchange and the other of the formation in solution of mixed complexes. This simplified theory clearly displays how the ammonia and copper(II) concentrations affect the amino acid fixation. Some experimental graphs illustrate the variation of the distribution coefficients for different amino acids with each of these parameters. Finally, some observations on the selectivity that may be expected from this process are made. In particular, it is shown how the optimal conditions for the separation of two amino acids can be determined.


Application of an ion-cluster technique to the analysis of endogenous hydroxylated compounds. I. Preparation of a doublet by use of a mixture of trimethylsilylimidazole (TSIM) and TSIM-d3.

In order to apply the intensity-matching technique to polyhydroxysteroids in biological fluids, the doublet of [M]+ and [M+3]+ was prepared in the process of derivatization by use of a mixture of trimethylsilylimidazole (TSIM) and TSIM-d3 and was used for mass fragmentography. The molar ratio of TSIM and TSIM-d3 was defined by the reciprocal of the number of hydroxyl groups in steroids. This technique may be useful in identifying low levels of the endogenous polyhydroxysteroids of interest in biological fluids.


Comparison of electron-capture and electrolytic conductivity detection for the gas-liquid chromatographic analysis of some perfluoro derivatives of four agricultural chemicals.

The gas chromatographic response of four perfluoro derivatives, of four agricultural chemical [diethylstilbestrol] (DES) clopidol, linuron and carbofuran] was examined. The derivatives varied in fluorine content from 3 to 30 atoms per molecule. The sensitivities of the derivatives were found to be ca. 10-100 times greater by electron-capture detection than by electrolytic conductivity detection (halogen mode). The sensitivity also was found to increase with increasing fluorine content for all derivatives by electrolytic conductivity, whereas by electron capture, DES and clopidol exhibited similar responses with either the penta-, hepta-, or pentadeca-fluoro derivatives. The sensitivity of linuron and carbofuran derivatives by electron capture varied with increasing fluorine content. For detection by electron capture, the hepta-fluorobutyryl derivatives were preferred for DES and clopidol, and either the hepta-fluorobutyryl or the pentadecafluorooctanoyl derivatives for linuron and carbofuran. For detection by electrolytic conductivity, the pentadecafluorooctanoyl derivatives were superior for all four compounds.


Protein binding of rifampicin to bovine serum albumin as measured by gel filtration.

A "large zone-small zone elution method" with Sephadex G-25 has been developed for the study of the interactions of [14C]rifampicin with bovine serum proteins. The method was demonstrated to be capable of yielding both qualitative and quantitative information about the binding reactions. However, its application requires a large amount of experimental work, and therefore it should be reserved for instances when other procedures are not applicable, e.g., with highly lipophilic ligands.


Concentration of headspace, airborne and aqueous volatiles on Chromosorb 105 for examination by gas chromatography and gas chromatography-mass spectrometry.

Techniques are described for the collection of volatile material from headspace vapours and the atmosphere and for the direct extraction of volatiles from aqueous solution by traps containing the porous polymer Chromosorb 105. The traps are inserted through a valve into a gas chromatograph which facilitates the desorption and transfer of the volatiles to high-resolution capillary columns. Selected applications of the technique are described.


Solid-phase radioimmunoassay of rubella virus immunoglobulin M antibodies: comparison with sucrose density gradient centrifugation test.

The solid-phase radioimmunoassay (RIA) method developed in our laboratory for demonstrating rubella virus-specific immunoglobulin G (IgG) antibodies (Kalimo et al., 1976) was further developed for demonstrating IgM antibodies. A total of 188 serum specimens were tested. The statistical probability of obtaining a false-positive IgM result, based on determinations of 100 rubella-negative sera, was below 0.001. Nonspecific inhibitors and IgM antibodies against other viruses tested did not interfere in the assay. In 2 out of 20 (10%) serum specimens with rheumatoid factor, a false-positive IgM result was obtained. The new RIA method was compared with sucrose density gradient centrifugation, followed by hemagglutination inhibition testing of the separated immunoglobulins with respect to demonstrating IgM antibodies. In patients with acute rubella infection, IgM antibodies were demonstrated by RIA in 9 out of 20 acute-phase sera and in all 20 early-convalescent-phase sera, compared with 7 out of 20 acute-phase sera and 19 out of 20 early-convalescent-phase sera by sucrose density gradient centrifugation. The results obtained indicate that the RIA method is reliable and sensitive and suitable for routine diagnostic use.


Single phage-typing set for differentiating salmonellae.

A phage-typing system is described for characterizing commonly isolated salmonellae. Fifty-eight serovars representative of groups A, B, C1, C2, D, E1, E2, E3, and E4 were delineated by using a single set of 50 phages isolated from sewage. All of the 735 cultures used in this effort were typable and were distinguished and differentiated on the basis of the 347 phage patterns observed. All results were reproducible. Characteristic phage patterns were produced by a variety of Salmonella serovars isolated from a campus incident and a number of hospital and family outbreaks to indicate an existing epidemiological relationship.


Yersinia enterocolitica: recovery and characterization of two unusual isolates from a case of acute enteritis.

Enteritis caused by Yersinia enterocolitica appears to be an uncommon occurrence in the United States. Most of the reported cases have been caused by biochemically typical Y. enterocolitica serotype O:8, the most frequently encountered serotype in the United States. The present report describes the isolation of two biochemically and serologically unusual Y. enterocolitica isolates from a patient with acute enteritis. One strain was distinguished by the rapid fermentation of rhamnose and raffinose and by citrate utilization at 22 degrees C but not at 37 degrees C. The other isolate was sucrose negative, and at either temperature it lacked both the fermentative capability for rhamnose and raffinose and the ability to utilize sodium citrate. Neither strain was agglutinable with known Y. enterocolitica antisera. The rhamnose-positive isolate showed an increased resistance to ampicillin, cephalothin, colymycin, and penicillin when tested at 22 degrees C as compared to results obtained at 37 degrees C. The demonstration that one patient's serum contained agglutinins (1:64) against the sucrose-negative strain supports its etiological significance. The role of the rhamnose-positive strain in the patient's illness is speculative. It conceivably could have potentiated the pathogenicity of the sucrose-negative isolate.


beta-Glucosidase activity in mycobacteria.

A procedure to test beta-glucosidase was developed for identification of the mycobacteria. One hundred and thirty-three strains representing 17 species were assayed in a buffered system containing whole cells and the substrate p-nitrophenyl-beta-D-glucoside. Qualitative differences between species indicated that the test was useful in discriminating among the mycobacteria.


Renal tubular effects of chronic phosphate depletion.

The effects of chronic phosphate depletion on renal tubular function were evaluated by micropuncture and free water clearance studies in the dog. Proximal tubular punctures demonstrated that chronic hypophosphatemia led to a reduction in ratio of tubular fluid to plasma inulin in late superficial tubular from 1.59+/-0.08 in control animals to 1.29+/-0.06 in phosphate-depleted dogs, with proportional inhibition of calcium and sodium reabsorption. The chronic decrease in proximal tubular fluid reabsorption was confirmed by the analysis of sustained water diuresis in conscious, phosphate-depleted dogs, before and after repletion of body PO4 stores, and in control animals. Urine flow rate/100 ml glomerular filtration rate (V/GFR) was significantly higher in PO4 DEPLETION THAN CONTROL (15.8+/-1.1 VS. 10.7+/-0.82). In addition, acetazolamide infusion did not increase V/GFR in phosphate-depleted dogs (15.8+/-1.1 vs. 17.16+/-0.9), supporting the conclusion that inhibition of proximal tubular fluid reabsorption was responsible for the elevated urine flow rate. PO4 repletion over 5 days reduced V/GFR to 9.2+/-0.7 despite no change in urine osmolality and no change in GFR, further suggesting a specific reversible alteration in proximal tubular reabsorption in phosphate depletion. Although hypercalciuria was a constant finding in phosphate depletion (fractional excretion of calcium of 2.04+/-0.4% vs. 0.47+/-0.13% in controls), the enhanced distal delivery of calcium was not a crucial factor; acute phosphate infusion reduced urinary calcium excretion to control values without affecting the reduced proximal tubular reabsorption in either intact or thyroparathyroidectomized phosphate-depleted dogs the change in distal nephron calcium reabsorption was independent of parathyroid hormone (PTH) levels since infusion of PTH failed to alter urinary calcium excretion. We conclude that chronic phosphate depletion leads to a reversible, sustained inhibition in proximal tubular reabsorptive fuction as well as a specific decrease in distal nephron calcium reabsorption. This latter reabsorptive defect is sensitive to phosplate infusion but not corrected by PTH.


Quantitative determination of antibody to capsular polysaccharide in infection with type III strains of group B Streptococcus.

The development of antibody in response to invasive infection with type III strains of group B Streptococcus was studied in sera from 31 infants and 4 adults by means of a quantitative radioactive antigen-binding assay. Low concentrations of antibody were consistently found in the acute sera of patients who developed clinical illness. Although adults with puerperal sepsis and infants with bone or joint infection uniformly demonstrated significant rises in serum antibody concentration after recovery, much lower levels of antibody were detected in convalescent sera from infants recovering from meningitis or sepsis. The median antibody concentration in sera from 43 parturients with type III strains of group B Streptococcus isolated from vaginal cultures whose neonates failed to develop symptomatic disease was significantly greater than that in sera from 29 mothers of infants with invasive, type III, group B streptococcal infection. Study of paired maternal and cord sera demonstrated a significant correlation between the antibody concentration in a mother's serum and that in her neonate.


Biliary lipid secretion in cholesterol gallstone disease. The effect of cholecystectomy and obesity.

Cholesterol gallstone disease is initiated in a liver which produces abnormal bile with excess cholesterol relative to bile salts and phospholipid. To define the responsible secretory mechanism(s), the rate of biliary lipid secretion was measured by a duodenal marker perfusion technique, while the bile salt pool was simultaneously estimated by isotope dilution. Two groups of control patients expected to have normal biliary lipid composition--14 subjects without hepatobiliary disease and 6 patients with pigment gallstones, were compared to two experimental groups expected to have abnormal bile--10 nonobese patients with cholesterol gallstones and 7 obese subjects without gallstones. Both control groups had nearly identical biliary lipid secretion rates, and a corresponding low relative molar concentration of cholesterol. Two different secretory mechanisms were found to be responsible for the abnormal bile in the experimental groups. In the nonobese patients with cholesterol gallstones, bile salt and phospholipid secretion rates were both significantly reduced. Conversely, the grossly obese subjects had an increased cholesterol secretion. To determine how cholecystectomy improves biliary lipid composition, three groups of gallstone patients --6 with pigment stones, 4 grossly obese with cholesterol stones, and 13 nonobese with cholesterol stones --were all examined after full recovery from surgery. In the nonobese patients with cholesterol gallstones, both bile salt and phospholipid secretion significantly increased, causing a definite improvement in bile composition. Cholecystectomy produced a similar but less marked trend in the obese patients with cholesterol stones, and in the patients with pigment stones. Cholesterol secretion, however, was unaffected by surgery. The bile salt pool was definitely small in the nonobese patients with cholesterol gallstones and became slightly smaller after cholecystectomy. The pool was significantly reduced by cholecystectomyin the patients with cholesterol gallstones. Removal of the gallbladder in all three groups caused a greater fraction of the pool to cycle around the enterohepatic circulation each hour. This more rapid cycling produced the increase in bile salt and phospolipid secretion, and was responsible for the improved composition found after cholecystectomy.


Influence of chronic renal failure on protein synthesis and albumin metabolism in rat liver.

Chronic renal failure in rats leads to changes in hepatic protein synthesis and albumin metabolism at both the cellular and molecular level. In rats with chronic uremia (blood urea nitrogen  greater than 45 mg/100 ml 1 mo after surgical reduction in renal mass), cell-free protein synthesis is reduced 30--40% in liver membrane-bound polyribosomes. Albumin synthesis by membrane-bound polysomes in uremia is reduced even more than the reduction in total protein synthesis. Activity of free polysomes remains norma. There is also intracellular accumulation of albumin in liver of uremic rats and a concomitant decrease in serum albumin. In normal liver, most intracellular albumin is located in the microsomal fraction, whereas in liver from uremic animals the excess albumin is found in the free cytosol fraction. These results can be explained either by a defect in synthesis of albumin by membrane-bound polysomes with release of newly synthesized albumin into the cytosol or by a reduced ability of polysomes synthesizing albumin to associate with the membrane fraction in rats with chronic renal failure.


Hemodialysis leukopenia. Pulmonary vascular leukostasis resulting from complement activation by dialyzer cellophane membranes.

Acute leukopenia occurs in all patients during the first hour of hemodialysis with cellophanemembrane equipment. This transient cytopenia specifically involves granulocytes and monocytes, cells which share plasma membrane reactivity towards activated complement components. The present studies document that complement is activated during exposure of plasma to dialyzer cellophane, and that upon reinfusion of this plasma into the venous circulation, granulocyte and monocyte entrapment in the pulmonary vasculature is induced. During early dialysis, conversion of both C3 and factor B can be demonstrated in plasma as it leaves the dialyzer. Moreover, simple incubation of human plasma with dialyzer cellophane causes conversion of C3 and factor B, accompanied by depletion of total hemolytic complement and C3 but sparing of hemolytic C1. Reinfusion of autologous, cellophane-incubated plasma into rabbits produces selective granulocytopenia and monocytopenia identical to that seen in dialyzed patients. Lungs from such animals reveal striking pulmonary vessel engorgement with granulocytes. The activated complement component(s) responsible for leukostasis has an approximate molecular weight of 7,000-20,000 daltons. Since it is generated in C2-deficient plasma and is associated with factor B conversion, it is suggested that activation of complement by dialysis is predominantly through the altermative pathway.


Factor IX antigen by radioimmunoassay. Abnormal factor IX protein in patients on warfarin therapy and with hemophilia B.

Factor IX, isolated from normal human plasma, was homogenous by polyacrylamide gel electrophoresis in urea and sodium dodecyl sulfate. On the latter, it migrated as a single polypeptide chain with or without reducing agents and had an apparent mol wt of 62,000. After iodination by chloramine-T, a single peak of 125I was found on gels. Immunoelectrophoresis in agarose with rabbit antifactor IX sera gave a single arc against both isolated and partially purified factor IX preparations. The rabbit antibody was specific as it failed to inhibit the activities of prothrombin or factors VII or X in normal plasma. At an additional 20-fold dilution, factor IX activity was inhibited 50%. In a double-antibody radioimmunoassay, excess rabbit anti-human factor IX precipitated 90-95% of the 125I-human factor IX. Control without specific antibody gave 6-8%. Dilutions of a pool of normal human plasma paralleled dilutions of the isolated preparation and were used for the standard curve. Of 39 plasma samples from normal donors, the mean factor IX antigen level was 93% of that of a separate normal pool. The radioimmunoassay detected the abnormal factor IX produced in patients on warfarin therapy. After Al(OH)3 adsorption of warfarin treated patient's plasma, factor IX antigen, but not activity, was present in the supernate. Samples from 28 patients on warfarin gave a mean factor IX clotting activity of 27% with a mean antigen of 69%. The antigen level from the warfarin group was significantly lower than the antigen level of the normal group (P less than 0.001). The factor IX antigen level was then assessed in 36 patients from 29 pedigrees with hemophilia B. The median antigen level was 17% of normal. The distribution of the antigen level was wide with two patients around 100% of normal; only two had levels below the limits of resolution of the radioimmunoassay as currently performed (less than 2%). Within each of the five pedigrees in which more than one affected member was tested, activity and antigen levels were the same. The degree of neutralization of the antibody's inhibition of normal plasma by patient's plasma was highly correlated. Additional evidence for the detection of abnormal protein was provided by immunodiffusion of plasmas concentrated by lyophilization. Reactions of complete identity occurred between normal, a warfarin treated and a hemophilia B subject's plasmas.


Jejunal perfusion of simple and conjugated folates in celiac sprue.

The intestinal absorption of [3H]-pteroylmonoglutamate (simple folic acid) and pteroyl-micron[14C]glutamyl-gamma-hexaglutamate ([14C]PG-7, conjugated folic acid) was assessed by the method of jejunal perfusion in five patients with proven celiac sprue who were studied after a gluten-containing or a gluten-free diet, and in nine normal subjects. The luminal disappearance of each folate was markedly impaired after exposure of the patients to dietary gluten and improved by gluten restriction, but not to within the range found in the normal subjects. The luminal disappearance of each folate was markedly impaired after exposure of the patients to dietary gluten and improved by gluten restriction, but not to within the range found in the normal subjects. In each experiment, column chromatography of the luminal aspirates revealed similar spectra of hydrolytic products of [14C]PG-7, whereas the fraction of the distal aspirate chromatogram appearing as pteroyl-micron[14C]glutamyl-gamma-monoglutamate ([14C]-PG-1) was similar in all three groups. By accounting for the variable effects of absorption on the luminal appearance of [14C]PG-1 and by correcting for mucosal hydrolysis which was not followed by release of [14C]PG-1 to the luminal contents, the calculated rate of in vivo hydrolysis of [14C]PG-7 to [14C]PG-1 was found impaired in both celiac sprue groups, with significant improvement on treatment. In mucosal biopsies from the sprue patients, the in vitro activity of folate conjugase in whole homogenates was higher and the activity of disaccharidase lower than in a group of 12 normal mucosal biopsies. These in vitro data suggest that the predominant cellular location of mucosal folate conjugase is different from that of disaccharidase, whereas comparison with the results of in vivo hydrolysis suggests that measurement of the enzyme in whole mucosal homogenates overestimates its significant digestive activity. The present studies indicate that (a) the mucosal lesion of celiac sprue significantly limits the intestinal absorption of both simple and conjugated folate, and (b) malabsorption of conjugated folate results from a combination of impaired hydrolysis and decreased mucosal uptake of hydrolytic product.


Calcium and phosphate transport in isolated segments of rabbit Henle's loop.

Calcium and phosphate transport was examined in rabbit thin descending, thin ascending, and thick ascending limbs of Henle by in vitro perfusion of isolated tubular segments. Permeability coefficients for these segments with 45Ca and 32PO4 were determined for both lumen-to-bath and bath-to-lumen directions. Both the thin descending and thin ascending limbs were found to be relatively impermeable to both 45Ca and 32PO4. In neither segment were we able to show evidence for net transport of calcium or phosphate. In contrast, the thick ascending limb of Henle showed a decrease in calcium lumen-to-bath concentration from 0.97 +/- 0.02 to 0.88 +/- 0.02 when perfused at 4.8 nl min-1. 45Ca lumen-to-bath and bath-to-lumen fluxes were 19.96 +/- 1.05 and 9.89 +/- 0.02 peq-min-1-cm-1, respectively, and the potential difference was +3.8 +/- 0.3 mV (lumen positive). The observed calcium flux ratio was significantly higher than that predicted by Ussing's equation. When ouabain was added to the bath the potential difference fell to +1.1 +/- 0.3 mV, whereas the calcium efflux was only slightly diminished (29.5 +/- 5.3-23.7 +/- 5.1 peq-cm-1-min-1). Ouabain had no effect on the influx of Ca across the thick ascending limb of Henle. There was no net transport of phosphate across the thick ascending limb. Phosphate permeability was exceedingly low bidirectionally across the thick ascending limb. Our findings indicate: (a) all segments of Henle's loop are relatively impermeable to calcium and phosphate; (b) net transport of phosphate seems to be absent in Henle's loop; (c) net calcium reabsorption, which cannot be explained by passive mechanisms, occurs in the thick ascending limb.


Circulating immune complexes in subacute infective endocarditis and post-streptococcal glomerulonephritis.

Two out of five patients with subacute infective endocarditis and two patients with post-streptococcal glomerulonephritis had large amounts of soluble circulating immune complexes in their sera. The three others with endocarditis had less severe disease and minimal evidence of circulating immune complexes. Low serum complement was also found in two of the cases.