Structural transitions of deoxyribonucleic acid in aqueous electrolyte solutions. II. The role of hydration.

The data and approach reported in paper I (Hanlon et al., 1975, preceding paper) have been used to calculate the fractional changes in secondary structure of calf thymus deoxyribonucleic acid which occur in aqueous solutions as a function of the concentration of NaCl, KCl, LiCl, CsCl, and NH4Cl. There is a continuous loss in the "B" character of the nucleic acid with concomitant production of the C and, in some instances, an A form, as well, as the salt concentration increases. Sedimentation velocity studies suggest that there is an accompanying change in the hydrodynamic characteristics of the DNA molecules, as well. Utilizing the existing hydration data in the literature (Hearst and Vinograd, 1961a,b; Hearst, 1965; Tunis and Hearst, 1968a; Cohen and Eisenberg, 1968; Falk et al., 1962, 1963a,b), we have found that a gradual loss of "B" character and a decrease in the frictional coefficient of DNA occur as the net hydration of DNA is reduced from the fully hydrated from (60-80 mol of H2O/mol of nucleotide) to values of ca. 12-14 mol of H2O/mol of nucleotide. Below that value, a more precipitous decrease in these properties occurs. Extrapolation of the linear relationship observed between the fractional B content and the net hydration in the latter regions yield  values of ca. 18 mol of H2O/mol of nucleotide at 100% B and ca. 4 mol of H2O/mol of nucleotide at 0% B (i.e., 100% C or C + A) for the alkali metal salts of DNA. The ammonium salt retains somewhat more H2O in the C and A forms (ca. 7). These results together with the hydration site assignments of Falk et al. (1962, 1963a,b) are interpreted in terms of a hydration model for DNA in aqueous solution in which an intact primary hydration shell of ca. 18 mol of H2O/mol of nucleotide is required for the maintenance of the "B" conformation. Removal of all but those water molecules solvating the phosphate groups results in the conversion to the C forms, predominantly, with a small amount of A structure formed as well in some salts. The accompanying changes in the sedimentation coefficients suggest that the DNA molecule assumes a more compact and/or flexible form under these conditions in which it is mainly in the C and A structures. The combination of these two events which ensue upon dehydration create a polymeric structure which can be more easily packaged in biological systems.


Electrophoretic microheterogeneity and subunit composition of the 13S coupling factors of oxidative and photosynthetic phosphorylation.

Two electrophoretically distinguishable species of the 13S coupling factor of oxidative phosphorylation from Alcaligenes faecalis are detectable by standard polyacrylamide gel electrophoresis in the absence of urea, detergents, or any other protein-denaturing reagents. The slower species (type IA) can be converted into the faster species (type IB) by treatment with ATP, and the fast form converts into the slow form when aged at 4 degrees. The enzyme undergoes these conversions both when it is free in solution and when it is membrane bound. The ATP analog adenylyl imidodiphosphate (AMP-PNP) gives the conversion without being hydrolyzed and without causing any apparent change in the mass of the protein, which suggests that the conversion may be a ligand-induced conformational change. Types IA and IB can convert into three other electrophoretically distinguishable species (types IIA, IIB, and III) if the purification procedure involves chromatography on a DEAE-Sephadex column equilibrated in phosphate buffer. These conversions can be prevented if the column is eluted in morpholinoethanesulfonic acid (Mes) buffer and KCl. Type IIA is convertible into type IIB by ATP treatment. Types IA and IB will also convert into types IIA and IIB and finally into type III when aged for extended periods of time at 4 degrees, without a detectable change in mass. Coupling factor activity is lost when type I enzyme converts into type II enzyme, as is the ability of the enzyme to bind to the membrane. However, ATPase activity does not change significantly. The mitochondrial 13S coupling factor shows up to three electrophoretically distinguishable species. The use of phosphate buffer during DEAE-Sephadex chromatography gives conversion of slower species into faster species. ATP treatment does not give interconversions, and aging at 4 degrees gives only a slow dissociation of the enzyme into subunits. The chloroplast 13S coupling factor also shows up to three electrophoretic species. Incubation with ATP does not give interconversions, but a temperature-dependent conversion of the major species into a faster species occurs upon aging. The subunit composition of the three 13S enzymes is very similar by polyacrylamide gel electrophoresis in sodium dodecyl sulfate, the major difference being in the number of classes of small polypeptides.


Immunoglobulin of T lymphoma cells. Biosynthesis, surface representation, and partial characterization.

Four cloned continuously cultured mouse T lymphoma cell lines, WEHI-22.1, WEHI-7.1, S49.1, and EL-4.1, were examined for immunoglobulin biosynthesis and the presence of immunoglobulin on the cell surface. Incorporation of [-3H]leucine into cellular proteins followed by serological analysis showed that immunoglobulin constituted between 0.1 and 1.1 percent of protein synthesized by the different cell lines during a 6-hr period. Under the same conditions cultured cells of nonlymphoid origin, the mastocytoma P-815 X-2.1, did not synthesize any detectable immunoglobulin. Lactoperoxidase-catalyzed radioiodination was used to label proteins on the surface of viable lymphoma and mastocytoma cells. Although the lymphoma lines lacked immunoglobulin as assessed by fluorescent antibody staining, immunoglobulin was detected in surface proteins of all four lymphoma lines. Estimates of the number of immunoglobulin molecules on the cell surface were 1.1 times 10-4/cell for S49.1 and EL-4.1, 1.7 times 10-4 for WEHI-7.1, and 4.3 times 10-4 for WEHI-22.1. Electrophoretic mobilities in sodium dodecyl sulfate polyacrylamide gel indicated that intact cell surface immunoglobulin was slightly larger than IgG, and on disulfide bond reduction to dissociate into two components, one with the mobility of serum immunoglobulin light chain, the other with a mobility similar to that of mu heavy chain. The heavy chain from the T lymphoma cells possessed an apparent molecular weight of about 65,000 compared with 70,000 for mu chain, although both chains shared antigenic determinants characteristic of mu chains. These findings are interpreted as support for other reports that T lymphocytes carry immunoglobulin on their surface and as direct evidence that thymus-derived lymphoid cells synthesize an immunoglobulin resembling the 7S subunit of IgM.


The stepwise binding of small molecules to proteins. Nuclear magnetic resonance and temperature jump studies of the binding of 4-(N-acetylaminoglucosyl)-N-acetylglucosamine to lysozyme.

The binding of 4-(N-acetylaminoglucosyl)-N-acetylglucosamine to lysozyme was studied by both nuclear magnetic resonance (NMR) and temperature-jump methods under comparable conditions. The NMR measurements on the inhibitor spectrum were carried out over a range of inhibitor concentrations including levels at which most of the inhibitor was bound to the enzyme. Data in this region were obtained by a novel difference method in conjunction with correlation spectroscopy. The results from the combination of both experimental techniques demonstrated the existence of a two-step binding mechanism and produced both values for all of the individual rate constants and also the NMR spectral data for the inhibitor in the two enzyme-inhibitor complexes. The later data characterize the environment experienced by the inhibitor at each stage in the binding process and thus provides both a three-dimensional and a dynamic picture of the interaction.


Catalytic properties of phospholipid exchange protein from bovine heart.

A protein which catalyzes the exchange of phosphatidylcholine between membranes has been purified from heart tissue homogenates up to 300-fold by acidic pH precipitation, (NH4)2SO4 precipitation, gel filtration, and ion-exchange chromatography. Binding of the protein to phosphatidylcholine liposomes as measured by Sepharose chromatography was nondetectable. However, isoelectric focusing experiments showed that individual molecules of phosphatidylcholine were transferred from liposomes to the soluble, partially purified protein. Exchange of phospholipid between liposomes and mitochondria was not affected by the presence of moderate amounts of cholesterol in liposomes. A search for competitive inhibitors among moieties similar to phosphatidylcholine failed to show strong binding sites in the hydrophilic part of the substrate. High concentrations of Na+, Ca2+ and Mg2+ impaired the exchange activity.


Preparation of erythrocyte ghosts by dielectric breakdown of the cell membrane.

Dielectric breakdown of membranes of red blood cells was observed in high electric fields (approx. 10-3-10-4 V/cm) using an improved Coulter Counter with hydrodynamic focussing. In making measurements of the size distributions of red blood cells as a function of increasing electric field strength it was found that a sharp discontinuity occurred in the otherwise linear relation between the pulse heights in the Coulter Counter and the electric field strength due to dielectric breakdown of the membranes. Solution of Laplace's equation for the electric field generated at breakdown in the cell membranes yeilds a mean value of about 1.6 V. for the membrane potential of red blood cells. Due to the dielectric break-down, release of hemoglobin occurred. Mechanical rupture of the red blood cells by the hydrodynamic forces in the orifice of the Coulter Counter or thermal rupture could be excluded as hemolysing mechanisms. The leaky ghost cells resealed at 37 degrees C. as shown by incorporation of 131I-labeled albumin and repeated dielctric breakdown.


Phospholipids in Newcastle Disease Virus infected cells.

Infection of chicken cells with Newcastle Disease Virus modifies phosphatidylserine and phosphatidylcholine synthesis in the host cell. The virion contains cellular phospholipids synthesized both before and after infection. Relative concentration of various labeled phospholipids in the virus differ from those in the corresponding cells and their surface membranes. Late in infection, fragments of membranes with a distribution of labeled phospholipids similar but not identical to that of the virus can be found in the supernatant of infected cells. The significance of these findings is discussed in relation to the origin of viral phospholipids and the intervention of the host cell membrane in the assembly of the viral envelope.


An evaluation of techniques for labelling the surface proteins of cultured mammalian cells.

We have evaluated four techniques for labelling the surface proteins of cultured mammalian cells. The techniques are: (a) the lactoperoxidase system; (b) the pyridoxal phosphate-[3H]borohydride system; (c) the [3H]4,4'-diisothiocyano-2,2'-dihydrostilbene disulfonate sysem and (d) the galactose oxidase-[3H]borohydride system. The subcellular distribution of radiolabel produced by these technics has been evaluated by autoradiography at the light microscope level and by cellular fractionation. We find that while all four systems label the surface membranes in the majority of the cell population, they also heavily label internal sites in a small subpopulation of nonviable cells. The contribution of the internally labelled cells to further biochemical analysis may represent a severe problem in investigations which rely solely on surface labels for the study of plasma membrane organization.


ATP- and phosphate-induced configurational changes of submitochondrial particles.

Light scattering was employed to monitor configurational changes of submitochondrial particles. Such changes were induced by ATP but not by analogues of this nucleotide. Mg-2+ in an equimolar concentration to ATP enhanced the effect of the nucleotide. The ATP-induced changes were inhibited by oligomycin and uncouplers. Atractyloside was effective as an inhibitor only when loaded within the particles. The ATP-induced changes were decreased by phosphate. The effect of phosphate was partially inhibited by mersalyl. Sodium phosphate and ammonium phosphate were more effective than potassium phosphate. The observed changes in light scattering were due to (a) events involved in energization and de-energization of the membrane, and (b) events concerning transport over the particulate membrane. The changes were specific for adenine nucleotides and phosphate.


Antimycin-insensitive mutants of Candida utilis II. The effects of antimycin on Cytochrome b.

1. Cytochrome b-562 is more reduced in submitochondrial particles of mutant 28 during the aerobic steady-state respiration with succinate than in particles of the wild type. When anaerobiosis is reached, the reduction of cytochrome b is preceded by a rapid reoxidation in the mutnat. A similar reoxidation is observed in the wild type in the present of low concentrations of antimycin. 2. In contrast to the wild type, inhibition of electron transport in the mutant has a much higher antimycin titre than effects on cytochromes b (viz., aerobic steady-state reduction; reduction in the presence of substrate, cyanide and oxygen; the 'red shift' and lowering of E'-o of cytochrome b-562). Moreover, the titration curve of electron transport is hyperbolic whereas the curves for the reduction are sigmoidal. The conclusion is, that in both mutant and wild type, the actions of antimycin on electron transport and cytochromes b are separable. 3. The red shift in the mutant is more extensive than in the wild type. 4. Cytochrome b-558 and cytochrome b-566 (that absorbs in mutant and wild type at 564.5 nm) do not respond simultaneously to addition of antimycin, indicating that they are two separate cytochromes. 5. The difference between the effect of antimycin on electron transport and cytochromes b reduction is also found in intact cells of the mutant. 6. A model is suggested for the wild-type respiratory chain in which (i) the cytochromes b lie, in an uncoupled system, out of the main electron-transfer chain, (ii) antimycin induces a conformation change in QH-2-cytochrome c reductase resulting in effects on cytochrome b and inhibition of electron transport, (iii) a second antimycin-binding site with low affinity to the antibiotic is present, capable of inhibiting electron transport.


Regulation of species-specific ribosomal RNA in a somatic hybrid cell.

When 13B hamster-mouse hybrid cells are harvested either right after 4 h of incubation with [me-3H]methionine or following 26 h of "chase" with excess non-radioactive methionine, in both cases about half of the labeled cytoplasmic rRNA is of hamster type. It had been previously shown in this laboratory (Eliceiri, G.L. (1973) Biochim. Biophys. Acta 312, 737-741) that when [3H]uridine was the radioactive precursor about 80% of the labeled cytoplasmic rRNA was of hamster type after a short incubation, and about half after a long incubation. It is postulated that a temporary difference in the specific acitivity of [3H]UTP in possibly segregated mouse and hamster types of nucleoli might account for these results. The master/mouse ratio of cytoplasmic rRNA in hybrid 13B is similar in free and in membrane-bound ribosomes, and in ribosomes of sparse (rapidly growing) cell populations and of confluent (slowly growing) cells.


Analysis of the fibrin-polymerizing reaction using sodium dodecylsulfate-agarose gel electrophoresis.

1. A new method of sodium dodecylsulfate gel electrophoresis, sodium dodecyl-sulfate-agarose gel electrophoresis, was developed. The new electrophoresis was shown to have a high and efficient resolving power for fibrin polymers and was also applicable to other proteins. 2. By sodium dodecylsulfate-agarose gel electrophoresis, the fibrin polymerizing reaction with activated fibrin stabilizing factor was analyzed in detail. The cross-linking between the gamma-chains was indicated to occur at an intermolecular level. The solubility of polymerized fibrin was suggested to be related mainly to the formation of the crosslinks between gamma-chains.


1-H and 13-C nuclear magnetic resonance spectra of the lipids in normal and SV 40 virus-transformed hamster embryo fibroblast membranes.

Well resolved 1-H and 13-C NMR spectra were obtained with normal and SV 40-transformed cell membranes. Estimation of the ratio of 13-CT2 values of the normal to transformed cell membranes showed an increased intermolecular motion in the transformed cell membranes. The temperature dependence of the (CH2) line in the 1-H spectra in the temperature range 298-343 degrees K shows an activation energy for the lateral diffusion of the fluid phospholipid regions in the normal cell membranes while the transformed ones show practically no temperature dependence in this temperature range. The fluidity of the phospholipid region in the transformed cell membrane seems to be significantly higher than that observed in the normal cell material. These data support and extend the findings concerning the mobility of the concanavalin A binding/agglutinating sites on the surface of normal and virus-transformed cells and suggest further approaches to the study of the membrane alterations in tumor cells.


The relationship between two murine DNA-dependent DNA polymerases from the cytosol and the low molecular weight DNA polymerase.

After aqueous subcellular fractionation and partial purification by phosphocellulose chromatography, murine cells are found to contain a low molecular weight DNA-dependent DNA polymerase (beta) in the nuclear fraction and two distinguishable DNA-dependent DNA polymerases (C-I and C-II) in the cytosol. Both C-I and C-II are found in testis, liver, and regenerating liver; the amount of C-I being several fold increased in the regenerating liver and in immature testis. C-I and C-II are distinguishable by the criteria of salt sensitivity, inhibition by single-stranded DNA, elution from phosphocellulose, inhibition by 0.3 mM N-ethylmaleimide, template preference, and sedimentation coefficient. C-II is dissociated by 0.25 M KC1 to an active form of DNA polymerase of sedimentation coefficient 3.5 S while C-I is not dissociated, maintaining its sedimentation coefficient of 7.2 S. Many similar chemical and physical properties of C-II and the low molecular weight nuclear DNA polymerase (beta) suggest that C-II may represent an aggregate state of beta monomers, The size, reaction properties and the increase in enzyme activity under conditions of rapid cellular proliferation suggest C-I is analogous to the alpha DNA polymerase.


Covalent linkage between RNA and nascent DNA in the slime mold, Physarum polycephalum.

When alpha--32 P-labeled deoxyribonucleoside triphosphates are injected into plasmodia of the eukaryotic slime mold, Physarum polycephalum, they are incorporated initially into strands of DNA which are mostly less than 300 nucleotides long. Sixty minutes after injection incorporated deoxyribonucleoside triphosphates are found in much longer strands. If the short strands found two minutes after injection are denatured and centrifuged to equilibrium in a Cs(2)SO(4) density gradient, they migrate to a density slightly greater than that of single-stranded Physarum DNA. When these short strands are treated with alkali to hydrolyze RNA, a small fraction of the incorporated -32P is made acid-soluble and is identified as a mixture of the four ribonucleoside 2',3'-monophosphates. Such transfer of -32P to ribonucleotides occurs when any of the 4 alpha--32P-labeled deoxyribonucleoside triphosphates is used for injection, but the transfer is greatest with [alpha--32P]dGTP. We conclude that very short stretches of RNA are found linked through phosphodiester bonds to nascent DNA chains in Physarum polycephalum and that any of the 16 possible combinations of ribo- and deoxyribonucleotides can occur at the RNA-DNA junction.


Solution of ribosomal proteins under mild conditions.

Ribosomal proteins from two eucaryotic species, prepared by either the guanidine . HCl or LiCl . urea method and subsequently dissolved in 8 M urea were found to be largely retained in solution after removal of the urea by dialysis against a solution of low ionic strength (0.05 M Tris . HCl, pH 7.6, 0.025 M KCl, 0.005 M magnesium acetate) and centrifugation at 100,000 times g. The protein composition of this preparation was virtually identical to that of the original urea-containing solution as determined by two-dimensional polyacrylamide gel electrophoresis. Thus, there exists a very simple method for obtaining the bulk of the ribosomal proteins in solution under conditions where ribosomes themselves are stable.


Nitrogenase. VI. Acetylene reduction assay: Dependence of nitrogen fixation estimates on component ratio and acetylene concentration.

Acetylene reduction, an assay for nitrogenase activity (nitrogen:(acceptor) oxidoreductase, EC 1.7.99.2), Is dependent on the ratio of the two protein components of nitrogenase as well as on C2H2 concentration. As the component I : component II ratio (based on activity) is increased, the C2H2 reduction : N2 fixation ratio decreases to a minimum of 3.4 and then increases. The minimum is found at a ratio near 1 : 1. At a component I : component II ratio of 20 : 1, the C2H2 reduction : N2 fixation ratio is 5.3. Acetylene exhibits substrate inhibition in assays for nitrogenase activity. Both the apparent Km and Ki for acetylene vary as a function of the relative concentrations of components I and II present in the assay. When the more labile component II is limiting in the assay and "saturating" levels of C2H2 (above 0.1 atm) are used, N2-fixation capacity may be greatly under-estimated.


Inhibitory effect of D-glucosamine on glycolysis in bovine retina.

1. The effects of glucosamine concentration on the size of the lactate pool, on the levels of ATP, ADP, AMP and on the radioactivity incorporation from [1-14-C] glucosamine into lactate, N-acetylglucosamine and glucosamine-6-P were studied using whole bovine retinas. 2. The radioactive lactate, evaluated in relation to glucosamine molarity, after a modest initial increase, diminishes significantly. On the contrary the N-acetyl [1-14-C] glucosamine, the [1-14-C] glucosamine-6-P and, consequently, also the [1-14-C] glucosamine-6-P/[-14-C] lactate ratio increase with glucosamine molarity. 3. The retinal content of ATP shows a modest increment after incubation with low concentrations of D-glucosamine (0.5--2.0 mM) and a remarkable fall at higher concentrations. 4. Using retinal homogenates D-glucosamine clearly lowers the lactate production from glucose, glucose-6-P and fructose-1, 6-P2. 5. D-Glucosamine acts as an inhibitor of retinal glyceraldehyde-3-P dehydrogenase and lactate dehydrogenase by decreasing the initial velocity of these reactions. 6. It is concluded that D-glucosamine causes a reduction in the lactate production, by inhibiting two enzymes of the glycolytic pathway: glyceraldehyde-3-P dehydrogenase and lactate dehydrogenase. The fall in the adenine nucleotides content is a consequence of a dephosphorylation of ATP for the phosphorylation of glucosamine without concomitant resynthesis of ATP "via glycolysis".


Optimal conditions for uptake of exogenous DNA by Chinese hamster lung cells deficient in hypoxanthine-guanine phosphoribosyltransferase.

Conditions were characterized for maximizing the uptake of exogenous mammalian cell DNA by hypoxanthine-guanine phosphoribosyltransferase-deficient Chinese hamster lung cells. Recipient cell cultures in an exponential growth phase were found to be more competent in taking up DNA than stationary cultures. Polyornithine enhanced the uptake of exogenous DNA more reproducibly and to a greater extent than did any of the other facilitators tested (DEAE-dextran, CaCl2, latex spheres, spermine, polylysine and polyarginine). Maximal DNA incorporation occurred when polyornithine and DNA were mixed together prior to inoculation. About 25-30% of the DNA inoculum became deoxyribonuclease-resistant in a typical experiment utilizing polyornithine as the facilitator. Both homologous and heterologous exogenous DNAs rapidly became associated with recipient cell nuclei: approximately 95% of the deoxyribonuclease-resistant donor DNA was nuclear-associated 15 min after inoculation.


Fe(III) and Cu(II) conalbumin visible circular dichroism spectra.

The single polypeptide chain of conalbumin strongly binds two Fe(III) or two Cu(II) ions to yield intense absorption in the visible region similar to that shown by the related protein transferrin. Comparison of the metal-ion-binding sites in the two proteins is made by exploiting the sensitivity to ligand geometry of circular dichroism (CD). For the Fe(III) proteins strong similarities of the CD spectra outweigh marginal differences. For Cu(II) conalbumin an additional negative extremum near 506 nm appears between two positive ones at 634 and 410 nm suggesting greater subtraction of oppositely signed CD components leading to lesser magnitudes for the two positive peaks than are found in Cu(II)-transferrin. The two Fe(III)-binding sites within conalbumin are compared by noting the strong similarities of the CD and MCD of proteins with Fe(III) in one site and Ga(III) in the other site, and vice versa, with the protein containing Fe(III) in both sites. Due to features of the amino acid sequences of the single protein chains, the four strong metal ion binding sites in conalbumin and transferrin cannot be identical in all particulars, yet CD spectra of their metal ion complexes are closely similar. From a study of model phenolate complexes and the wavelength maxima of visible absorption in the Fe(III), Cu(II), and Co(III) proteins near 465, 440, and 405 nm, respectively, these strong absorption bands are identified as ligand to metal ion electron-transfer transitions. It is suggested that tyrosyl residues are the donors in the electron transfer transitions and that they lock in the metal ions after being keyed into position by binding of bicarbonate or other anions.


Platelet migration inhibition: a new method for detection of platelet antibodies.

A test for platelet antibodies based on the inhibition of migration of platelets from a capillary tube is described. The technique is simple and is capable of detecting antibodies directed against HL-A antigens and the platelet-specific antigen, Pl-A1. Inhibitory activity was detected in sera from two of seven patients with idiopathic thrombocytopenic purpura. The platelet migration inhibition test was as sensitive as platelet lysis and more sensitive than complement fixation, aggregometry, and platelet factor-3 release for detection of the antibodies studied.


Reaction of plasmic degradation products of fibrinogen in the radioimmunoassay of human fibrinopeptide A.

A radioimmunoassay (RIA) technique has been devised for the measurement of human fibrinopeptide A (FPA). The system utilizes rabbit antiserum to native human FPA and a synthetic fibrinopeptide, with tyrosine substituted for phenylanine in amino acid position 8. The test detects native human FPA at a concentration of 0.1 ng/ml, but does not cross react with human fibrinopeptide B or with fibrinopeptides A from canine, porcine, or bovine fibrinogen. Fibrinogen and chemical or plasmic degradation products with 2 moled of FPA per mole react fully in this test system. This includes the large-molecular-weight intermediate fragments X and Y and the NH2-terminal disulfide knot, and indicates that this antibody recognizes and reacts with FPA in the presence of the contiguous peptide structures present in fibrinogen. Fragment E, which is derived from the NH-2-terminal  portion of fibrinogen, loses most of its FPA content after its liberation from its precursor derivative and reacts to a lesser extent in the RIA than do fragments X and Y. This correlated with the recovery of FPA-positive material from ultrafilitrates of extensive but not partial plasmic digests of fibrinogen. Although FPA immunoreactivity liberated from fibrinogen does not necessarily reflect thrombin activity and/or fibrin formation, only extensive plasmic degradation yields peptide material which reacts in this RIA system. This should not be a serious limitation to the application of the RIA in the detection of venous thrombosis.


Sensitivity of spinal cords from chick embroys in culture to organic mercury compounds: comparison with dorasal root ganglia.

The lowest doses for total inhibition of MMC, EMC, EMP, and PMC on spinal cords from chick embryos in tissue culture were 2.5, 1, 5, and 2.5 times 10-6 M, respectively. From these results, it was presumed that spinal cord was more sensitive than dorsal root ganglion to these organic mercury compounds.


Effects of adrenaline, noradrenaline, isoprenaline and salbutamol on the production and release of renin by isolated renal cortical cells of the cat.

1 Isolated renal cortical cells of the cat have been demonstrated to produce renin on incubation in vitro. After 2 h of incubation, without added agonist, the total amount of renin in the flask increased by a mean of 27.2 percent. The increase in renin content of the incubation flask was found to be present in the medium. 2 Noradrenaline (1.18 times 10-minus 4M) and adrenaline (1.09 times 10-minus 4M) added to the incubation medium stimulated renin production by 45 and 34% respectively, compared with the incubated controls. Most of the increase in renin production was present in the incubation medium. 3 Isoprenaline did not stimulate renin production. However, when added to the incubation medium at a concentration of 0.72 times 10-minus 4M there was a significant decrease in the cellular content and a significant increase in the medium content of renin. This increase was at least as great as that observed with adrenaline and noradrenaline. 4 Salbutamol had an effect similar to isoprenaline, i.e. it induced the release of renin into the medium without affecting production. In this respect it was about a third as potent as isoprenaline.


Plasma nicotine levels after smoking cigarettes with high, medium, and low nicotine yields.

Plasma nicotine three minutes after smoking a cigarette was measured in 10 sedentary workers in mid-morning and five hours later on four typical working days. The average mid-morning level after they had been smoking their usual cigarettes (mean nicotine yield 1-34 ng) was 150-4 nmol/l (24-4 ng/ml) (range 95-6-236-7 nmol/l (15-5-38-4 ng/ml)). Despite great variation between smokersthe mid-morning levels of each smoker were fairly consistent over the four mornings and correlated 0-82 with their carboxyhaemoglobin levels. After continuing to smoke their usual brand or switching to a high-nicotine brand (3-2 mg) average afternoon levels of 185-6 and 180-0 nmol/6 (30-1 and 29-2 ng/ml) respectively were not significantly higher than the morning levels, but after switching to low-nicotine cigarettes (0-14 mg) the plasma nicotine dropped to an average of 52-4 nmol/l (8-5 ng/ml). The changes between morning and afternoon while smoking usual or high-nicotine cigarettes showed marked individual variation. The findings suggest that the plasma nicotine level just after a cigarette depends more on the way the cigarette is smoked than on its nicotine yield or the number which have been smoked over the preceding few hours.


Body fat of British and Dutch infants.

The fatty acids in the body fat of 41 British and 37 Dutch infants between birth and 1 year were determined. At birth linoleic acid contributed 1-3% of the total fatty acids of the body fat in infants in both countries. By one month its proportion in the fat of the Dutch infants was about 25% and by four months 32-37%; in the fat of the British infants it was never more than 3%. In the Dutch infants this large increase in the linoleic acid percentage was accompanied by a fall in the percentage contribution of others, particularly the saturated acids myristic, palmitic, and stearic. Infants born preterm showed changes in their fat after birth similar to those in fullterm infants. The difference between the composition of the fat of the infants in the two countries is attributed to the nature of the fat in the milk they received. Until recently most British infants who are not breast-fed have been given milks based on cow's milk with only minor modifications. For the past 10 years many Dutch infants have been given a milk in which all the cow's milk fat has been replaced by maize oil. Dutch infants also had a lower concentration of cholesterol in their serum than British infants, which was not unexpected. The results show that the triglycerides in the adipose tissue are profoundly influenced by the nature of the fat in the diet.


The effect of a new antibiotic, cryptosporiopsin, on RNA synthesis in L-cells.

The effect of cryptosporiopsin on RNA synthesis in L-cells was studied as part of an investigation on the mechanism of action and potential toxicity of the antibiotic in mammalian cells. RNA synthesis in vitro was tested in intact isolated L-cell nuclei, in conjunction with selective inhibitors of nucleolar and nucleoplasmic RNA synthetic activities; It was found that only the nucleoplasmic activity (polymerase II), was inhibited by cryptosporiopsin and that the drug showed no effect on the activity of the nucleolar enzyme (polymerase I). RNA synthesis in vivo was tested using double labelling with I114-C]guanine and [3-H]-uridine in an attempt at discriminating between G+C nucleolar trna and high A+U nucleoplasmic RNA synthesis. Results revealed that the uptake of these precursors into both types of RNA was inhibited by cryptosporiopsin in intact cells. Measurements of the nucleotide pools in these cells indicated that the antibiotic affects uptak and phosphorylation of nucleosides and nucleotides, especially the production of ATP; These results suggest that the uptake inhibition observed in vivo could be due, at least in part, to energy and/or precursor shortage.


Lysolecithin and cholesterol interact stoichiometrically forming bimolecular lamellar structures in the presence of excess water, of lysolecithin or cholesterol.

The structural interaction of egg lysolecithin, derived from egg lecithin, and cholesterol in aqueous solution has been investigated using X-ray diffraction. When mixed in any proportions, either suspended in excess buffer or up to 85% lipid by dry weight, a separate lamellar phase containing equimolar proportions of lysolecithin and cholesterol forms, separate from excess water, or lysolecithin or cholesterol. The cholesterol disorders the crystalline chains of the lysolecithin. The equimolar phase is stable up to 50 degrees C unlike lysolecithin alone, which forms micelles, Thes results show that lysolecithin and cholesterol combine stoichiometrically in a stable complex. We propose as a structural model, that cholesterol fills the space of the missing fatty acyl chain making the lysolecithin more cylindrical rather than wedge shaped. This interaction could reduce both the lytic action of lysolecithin on membranes and its induction of cell fusion. It suggest another role of cholesterol in cell membranes: namely, to act as a stabilizer of bilayer structure by being a mobile component that can fill free volume in the hydrocarbon interior. Lysolecithin-cholesterol interaction may also be important in the early events of atherosclerosis where lysolecithin levels in vessel walls increase fivefold.


Apomorphine: sterotyped behavior and regional distribution in rat brain.

The distribution of apomorphine following subcutaneous injection of 20 mg/kg (as the hydrochloride) was measured spectrofluorometrically in specific regions of rat brain. Measurable concentrations were found in the brain within a few minutes of injection, the drug was still detectable for at least 60 min in all regions, and maximum concentration was observed 20 min after injection. Stereotyped behavior, characteristic of apomorphine action, followed a time course parallel to accumulation of the drug in the brain.


Purification and some properties of the soluble and membrane-bound adenosine deaminases of Micrococcus sodonensis ATCC 11880 and their distribution within the family Micrococcacea.

In Micrococcus sodonensis and some other Micrococcus species, adenosien deaminase is present both as a membran-bound and a soluble enzyme; The membran-bound adenosine deaminase can be extracted with n-butanol, and may account for up to 5% of the total cellular adenosine deaminase activity. In a number oc comparative tests, no differences between the two enzyme forms could be found, thus they are believed to be similar molecular species; The purified membran-bound or soluble enzyme had a molecular weight, obtained by gel-filtration, of 130 000 and was inactive toward adenine and adenine mononucleotides. It appears, therefore, to be more closely related to the calf-intestine enzyme than the Aspergillus oryzae form in respect to size and substrate specificity; Attempts to correlate membrane-bound adenosine deaminase activity with adenosine transport in isolated membrane vesicles of M. sodonensis indicated no obvious relationship between the two activities.


Effects of changes in environmental lighting upon levels of hydroxyindole O-methyltransferase activity in the developing-chick gland.

The level of hydroxyindole O-methyltransferase (HIOMT) activity in the pienal gland of developing chicks raised under constant illumination rose more rapidly and to higher values than in the gland of birds maintained in constant darkness. Rates of net increase in activity, and levels of activity attained, for birds raised under a diurnal cycle of illumination were intermediate between those maintained in constant light or darkness. Under each of the lighting conditions, the course of increase in enzymic activity was markedly affected by variations in an unidentified factor, the source of which appeared to be the hatching eggs. Birds transferred from constant light to the dark showed either an arrest of increase in enzyme activity or a loss of activity until the levels equalled that observed for chicks of the same age raised in constant darkness. Chicks transferred from constant darkness to constant illumination showed marked increases in levels of enzyme activity at rates comparable with the maximal values observed with birds maintained under constant illumination, regardless of age and without delay. No diurnal cycle in level of HIOMT activity was observed in the pineals of 15-day birds.


Antibody production in goat milk serum after virus instillation of goat mammary gland. VII. Biochemical studies on sham infection with rabies and other non-propagatingagents.

This report describes the development of a method for the production and isolation of neutralizing antibodies against rabies virus (ERA strain) and other agents in the goat mammary gland during active lactation. The rabies virus did not propagate in thegland, but neutralizing antibodies were produced by serial instillations of the antigen. This process was termed 'sham infection.' Antibodies first appeared in the mildabout the 20th day and the titer increased until the 28th day. The antibodies were found to be associated with the lactogammaglobulin. The milk globulins were fractionated by gel chromatography and the fraction containing the antibody was isolated. This fraction was further purified and characterized by immunodiffusion, immunoelectrophoresis, and an analytical ultracentrifugation technique. The neutralizing antibody activityafter the 29th day was associated with the milk serum IgG fraction. This acive fraction was found to have a sedimentation coefficient of 6.8 Svedberg units.


Germ-cell malignant tumours in father and son.

Germ-cell malignant tumours occurred in a man and his son. The father, who had a teratoma of the right testicle removed 24 years ago, is presently alive and well. The son, who had a choriocarcinoma presenting as an abdominal mass, possibly originating in the testicle, died within 7 months of the diagnosis with metastases in the lungs, liver and retroperitoneum. This report documents the third such case of germ-cell neoplasms occurring in father and son.


Locomotion studies as an aid in clinical assessment of childhood gait.

A clinical locomotion laboratory has been developed to provide quantitative information in the management of gait disorders. The biomedical engineering development of this system identified two major clinical constraints: (a) the need for instrumentation that would not alter the natural gait of the patient and (b) the need for data-processing techniques that would permit analysis and correlation of the large volume of electromyographic (EMg) and kinematic information. The net result has been a unit that incorporates a multichannel telemetry system to capture the EMG and foot-switch information and a television computer system to handle the kinematic information. Gait studies on children with hemiparesis, muscular dystrophy and cerebral palsy have yielded quantitative EMG and kinematic information on the pathomechanics of ambulation in these disorders. Because the information obtained is quantitative, an accurate measure of improvement (or lack of it) after treatment can be documented. Therefore, the locomotion laboratory may have an important role in the preoperative and postoperative evaluation of children whose abnormal gait may require surgical corrective procedures or rehabilitative treatment including the use of prostheses or orthoses.


The future of child psychiatry. Mandate for change.

It seems that the future of child psychiatry is uncertain, since its present body of knowledge and its identity both within its own discipline and relative to others are unclear. Nevertheless, the vast problems of childhood and adolescence delineated by the recent plethora of reports indicate the unmet needs, and it is obviously necessary that child psychiatrists should join other groups in attempting to radically alleviate these problems. But although it has been fashionable to describe the radical child psychiatrist as liberal, flexible, non-committed and open, true radicalism in chid psychiatry would be signalled by a return to structure, by careful and circumscribed clinical activity and by a proper measure of humility.


Identical twins concordant for anorexia nervosa. A preliminary case report.

This case described the symptoms of anorexia nervosa occurring in identical twins. The problems of twinship, especially the failure to establish separate identities, complicated the more general psychological issues facing these adolescents in a disturbed family. In therapy the core issues of the anorexic patient persist--disturbance of body image, misinterpretation of body stimuli and a sense of ineffectiveness. Up to now therapy seems to have improved family function, making it easier for them to coexist and decreasing the feelings of hostility and negativism felt between the parents and the twins. Eating behaviour presently focuses on the twins' need to have equal amounts of food but less emphasis is placed on the food as an area of conflict for the family.


Inhibition of the metabolism of amino sugars with 2-deoxy-2-(2-fluoroacetamido)-alpha-D-glucopyranose.

The metabolism of amino sugars in extracts of mouse cells was studied by the use of 2-acetamido-2-deoxy-alpha-D-glucopyranose, 2-acetamido-2-deoxy-alpha-D-manno-pyranose, and N-acetylneuraminic acid as tracers with or without added 0.5mM 2-deoxy-2-(2-fluoroacetamido)-alpha-D-glucopyranosyl pyrophosphate) and cytidine 5'-(N-acetylneuraminyl monophosphate) was inhibited by the fluoro sugar.


A compartmental analysis of circulatory lymphocytes in the spleen.

A tentative model describing the passage of circulatory lymphocytes through the spleen is formulated in accord with known anatomical features. In order to preserve isomorphism between the model and the splenic system, the model is formulated in compartmental form and its design allows alternative routes and modes of lymphocyte transit to be considered. The simultaneous differential equations arising from the model are solved using an analogue computer which also provides the means whereby the performance of the model may be compared with suitable dynamic data drawn from literature. This not only allows the selection of a particular configuration of the model in preference to its alternatives, but also allows the numerical determination of certain unknown parameters. In the case of the rat spleen, best agreement between model and experimental data is obtained when between 10 and 25% of the total lymphocyte flux in the model spleen passes through the marginal zone where the average dwell time of the lymphocytes is about 50 min. The white pulp receives a lymphocyte flux from the marginal zone amounting to about 10% of the total splenic flux and the white pulp lymphocytes are sequestered for a period of 4-6 hr before release to the venous circulation. The red pulp receives 90% of the total splenic flux but the majority of lymphocytes find transit through the red pulp in less than 5 min. The remaining flux of lymphocytes, amounting to 10% of the splenic input, is delayed in transit through the red pulp by 2-3 hr before release to the venous circulation.


A kinetic estimation of base sequence complexity of nuclear poly(A)-containing RNA in mouse Friend cells.

Complementary DNA (cDNA) has been transcribed by viral reverse transcriptase from poly(A)-containing nuclear RNA prepared from growing mouse Friend cells (clone M2). Annealing experiments with this cDNA have demonstrated that a large proportion of the poly(A) tracts in M2 cell nuclear RNA are adjacent to RNA sequences which are transcribed from nonrepetitive DNA in the mouse genome. The kinetics of hybridization of cDNA to template RNA indicate that nuclear poly(A)-containing RNA consists of at least two abundance classes, the more complex of which is transcribed from approximately 3 percent of the genome. Thus there are at least 5 times more unique DNA sequences represented in nuclear polyadenylated RNA than in polysomal polyadenylated RNA. Moreover, there is evidence for posttranscriptional mechanisms which alter the relative concentrations of some (at least) gene transcripts between nucleus and cytoplasm.


Alpha-amanitin resistance: a dominant mutation in CHO cells.

Hybrids of CHO cells were constructed consisting of either a 1:1 or 1:2 ratio of alpha-amanitin-resistant and sensitive cells, respectively. The resistance of such hybrids to killing by the drug was similar but slightly less than that of the resistant parent. The hybrids contained both resistant and wild-type RNA polymerase II, in amounts related to the expected gene dosage. The alpha-amanitin marker therefore is expressed codominantly.


Polyribosomal structures inactive for protein synthesis present in erythroid cells during terminal stages of differentiation.

During the terminal stages of differentiation nucleated erythroid cells from the fetal mouse synthesize hemoglobin at a lower rate because after the last cycle of cell division about half of their polyribosomal structures are rendered inactive for protien synthesis though they maintain their aggregated shape. Partially inactive polyribosomes are tested in comparison with normal polyribosomes for the capacity to support polypeptide chain synthesis in cell-free conditions. The following observations are made: a) no difference is found for the profile on sucrose density gradients; b) partially inactive polyribosomes carry growing polypeptide chains in reduced amounts in comparison with active polyribosomes; c) partially inactive polyribosomes are not capable to release "run off" 80 S ribosomal monomers and to dissociate to active ribosomal subunits. These data are interpreted as the evidence for a block of chain termination producing inactivation of polyribosomes during the late maturation of nucleated erythroid cells.


PMR relaxation times of micelles of the nonionic surfactant Triton X-100 and mixed micelles with phospholipids.

Previous pmr studies at 220 MHz have led to the suggestion that phosphatidylcholine and the nonionic surfactant Trition-X-100 form mixed micellar structures at high molar ratios of trition to phosphalipid. These mixed micelles provide one form of the phospholipid which the enzyme phospholipase A2 can utilize as substrate. Spin-lattice relaxation times (T1) and spin-spin relaxation times (T2) obtained from line widths for resolvable protons in Triton X-100 micelles and mixed micelles with egg phosphatidycholine and dipalmitoyl phosphatidylcholine are reported. They suggest that the structure of the mixed micelles is generally similar to that of pure Triton X-100 micelles. The T1 values for the phsopholipid in the mixed micelles are found to be similar to those reported for phospholipid in sonicated vesicle preparations which are used as membrane models, but the lines are somewhat sharper suggesting the possibility of less anisotropic motion in the mixed micelles than in the vesicles.


A rapid and specific method for the determination of oxytocinase (cystine aminopeptidase) activity using continuous flow analysis and S-benzyl-L-cysteine-p-nitroanilide as substrate.

1. An automated method is described for the assay of pregnancy serum cystine aminopeptidase (Oxytocinase, E.C. 3.4.1.10) using continuous flow analysis. S-Genzyl-L-cysteine-p-nitroanilide, the most highly specific substrate so far available for oxytocinase is employed in this assay. Comparison with the manual method from which it has been developed, gave a correlation coefficient of 0.98. Reaction of oxytocinase on the substrate liberated p-nitroaniline, the absorption of which is measured at 410nm. 2. Blank values are eliminated by the incorporation of a dialyzer in the manifold design and thereby effectively increasing the rate of assay sample to 40 per hour. The lag phase (a) of the continuous flow analysis is 0.17 min while the exponential phase (b) is 0.31 min. Using a 2:1 sample to wash ration the carry-over is 3% at a 95% of steady states.


A rapid radioimmunoassay method for plasma luteinizing hormone.

1. A rapid double antibody radioummunoassay method for the determination of plasma luteinizing hormone (hLH) is described. 2. The method involves preincubation of the anti-LH serum with the precipitating second antibody and incubation of this mixture with unknows and standards at room temperature overnight. The incubation tubes are then centrifuged, supernatants decanted and the precipitates counted. 3. A total of two working days is required to complete the assay and report the results. 4. This method is more rapid than the conventional ones currently used for the estimation of LH in blood.


Estimation of plasma testosterone without extraction and chromatography.

A new radioimmunoassay for plasma testosterone in human blood plasma is described. The assay is performed directly upon the plasma without extraction and purification of the testosterone. Antibodies to testosterone-7alpha-carboxyethyl thioether-bovine serum albumin are used and data concerning the specificity of these antibodies are given.


Serum testosterone determination by mass fragmentography.

A mass fragmentographic reference method for the determination of plasma or serum testosterone is described. A fixed amount of [4--14C]testosterone (usually 10 ng) is added to a fixed amount of serum (usually 1 ml) and extracted with ether. The ether extract is purified by means of thin-layer chromatography. The purified testosterone is converted into the di-trimethylsilyl derivative by treatment with trimethylsilylimidazole. The amount of unlabeled testosterone is determined from the ratio between the recordings at m/e 432 and 434, obtained after analysis with a combined gas chromatograph-mass spectrometer equipped with a MID-unit (multiple ion detector). The two ions used correspond to the molecular peak in the mass spectrum of unlabeled and 4--14C-labeled testosterone, respectively. The relative standard deviation of the method was about 2.7 percent. The method was compared with a radioimmunoassay technique. There was a good correlation and the regression coefficient was about 0.83. A diurnal rhythm in testosterone secretion was confirmed both with the mass fragmentographic technique and with radioimmunoassay.


Preparation of high-quality iodine-125-labelled pituitary luteinizing hormone for radioimmunoassay.

High quality pituitary luteinizing hormone labelled with 125-I was obtained after separating out the more heavily iodinated fractions, through starch gel electrophoresis, using the cathodal component (Fraction 1) which was further purified on Sephadex G-100, giving an almost pure 125-I-labelled luteinizing hormone preparation, presenting excellent immunoreactivity and low levels of damage on incubation in plasma. The quality control of the steps of the technique was done with plasma-coated talc (200 mg) which compared favorably, as far as being an indication of intact labelled luteinizing hormone, with the more time-consuming chromatoelectrophoresis.


The magnesium load test: I. A design for infants.

The purpose of this study was to design the shortest suitable magnesium load test for infants up to six months of age. A 56-hour test was used to study cation and creatinine excretion before and after an intramuscular load of 0.49 mEq of magnesium/kg of body weight. No diurnal excretion pattern for magnesium was found, and most of the magnesium rejected by the kidney was excreted by neonates within 32 hours, and by infants from one to six months of age within 24 hours. The postload urinary magnesium value usually stabilized at a slightlyhigher level than the preload level. The mean plasma magnesium level increased about 0.65 mEq/liter one hour after the load, and at the end of the test was 0.25 mEq/liter higher than the initial value; abnormally low plasma values were normalized. Renal excretion of creatinine and potassium was not affected by the magnesium load, but the 24-hour postload excretion was higher than the 24-hour preload excretion of calcium for all patients, and of sodium for some full-term neonates. Because of slow excretion of magnesium and concomitant caocium loss, special precautions are required for testing and repletion of young infants to avoid magnesium overload and calcium depletion. A suitable test would have an eight-hour preload period, a load of 0.49 mEq of magnesium (0.12 ml of 50 per cent MgSO4.7H20) per kg of body weight, and a postload collection of 32 hours for neonates and 24 hours for infants from one to six months of age.


The characteristics of ferritin from human tissues, serum and blood cells.

1. The properties of ferritin in serum have been compared with those of ferritin from a number of tissues including blood cells. On anion-exchange chromatography with DEAE-Sephadex, the behaviour of human heart ferritin is different from that of liver, kidney or spleen ferritin. Reticulocyte ferritin appears to have similar characteristics to heart ferritin. 2. Serum ferritin from normal subjects and patients with various degrees of iron load, leukaemia or liver disease all have a much lower affinity for the anion-exchange column that any tissue ferritin, suggesting a difference in isoelectric point. The elution point of serum ferritin from patients with acute myeloblastic leukaemia is significantly different from normal. 3. Density gradient centrifugation in sucrose showed that ferritin in leucocyte extracts and partially purified ferritin from the serum of two patients with iron overload behaved as apoferritin rather than the iron-rich protein. 4. The results suggest that ferritin is modified during its entry into the plasma and that even in cases of iron overload the iron content of serum ferritin may be low. The findings are of importance in considering the origin of plasma ferritin, the clearance of ferritin from plasma and its role in iron metabolism.


Impaired hepatic release of triglycerides: a possible cause of acute alcoholic fatty liver in the Rhesus monkey.

1. The mechanism of development of acute alcoholic fatty liver in the Rhesus monkey was investigated by studying the effect of Triton WR-1339 on plasma triglycerides. 2. After ethanol infusion, the rise in plasma triglyceride produced by Triton was greatly reduced and there was an accumulation of triglyceride in the liver. 3. Thus findings suggest that acute alcoholic fatty liver in Rhesus monkeys is probably due to a defect in the release of triglyceride from the liver.


Isolated anomalous right ventricular muscle bundle in the asymptomatic adult.

Muscular obstruction to right ventricular outflow is usually at the infundibular level, but, less commonly, it can occur within the body of the ventricle due to an anomalous muscle bundle. This lesion is almost always associated with a ventricular septal defect or other cardiac lesions, and is usually discovered in infancy or childhood. Two asymptomatic adults with severe obstruction due to right ventricular muscular band as an isolated lesion are presented. The lesion can be tentatively diagnosed on clinical grounds and confirmed by angiocardiography. The advisability of surgical correction in the asymptomatic adult with such an isolated lesion is uncertain.


Testicular maintenance and masculinized development of the vagina of the androgen-insensitive male rat pseudohermaphrodite.

The vaginae of about one-third of androgen-insensitive male rat pseudohermaphrodites never open nor can they be opened by the administration of estrogen, progesterone or andorgens. The vaginae of the remaining pseudohermaphrodites open significantly later than normal females, and, once open, require the presence of the testes or exogenous estrogen to maintain their patency. These defects in the vaginal development of the pseudohermaphrodite may be due to some perinatal masculinization of the perineum, unlike most of the phenotypically female development exhibited by this animal.


LH effect on the pattern of steroidogenesis in cultured Graafian follicles of the rat: dependence on macromolecular synthesis.

Graafian follicles explanted from proestrous rats before the preovulatory gonadotropin surge secreted predominantly 17beta-estradiol, and only small amounts of progestins and androgens, during 12 h of culture in hormone-free medium. Addition of ovine luteinizing hormone (NIH-LH-S18; 0.1-1.0 mug/ml) to the medium stimulated within 1-2 h the rate of accumulation of these steroids. However, accumulation of androstenedione and estradiol ceased after 4-6 h in the LH-treated cultures, whereas progesterone continued to accumulate and became the major secretory product at 6-12 h. Incubation of the follicles with LH for only 5 min resulted in significant stimulation of the accumulation of progesterone, androstenedione and estradiol during a subsequent 6-h culture period in hormone-free medium containing antibodies to LH; 30 min exposure to the hormone sufficed to elicit a maximal steroidogenic response and to induce ovum maturation in 95% of the follicles. Addition of actinomycin D (act D; 8 mug/ml) within the first hour after exposure of follicles to LH suppressed the LH-effect on progesterone but augmented the LH effect on estradiol accumulation; when addition of this inhibitor was delayed until 2 h, progesterone accumulation continued unabated for a further 10 h. By contrast, puromycin (80 mug/ml) inhibited progesterone accumulation when added to the medium at any time (1, 2 or 3 h) after the hormone. It is suggested that (i) a short-lived protein is essential for the stimulatory effect of LH on follicular steroidogenesis; (ii) the act D-sensitive product (mRNA?) required for the production of this protein is synthesized in adequate amount during the first 2 h of LH action, and is stable for at least 10 h; and (iii) LH may stimulate the production of an additional act D-sensitive regulatory protein that inhibits enzymes engaged in the cleavage of the 17-side-chain of progesterone, or cells equipped with these enzymes.


Comparsion of hydroxyindole-O-methyltransferse levels in pineal glands of normal, blinded, and congenitally anophthalmic guiena pigs.

The level of pineal hydroxyindole-O-methyltransferase activity was measured in normal sighted, normal blinded, and congenitally anophthalmic guinea pigs at 1100-1200 and 2300-2400 h. Significant day-night differences were found in the normal sighted and normal blinded guinea pigs; however, the levels were markedly elevated in the normal blinded guinea pigs. The congenitally anophthalmic guinea pigs did not demonstrate a difference between day and night activities. Both the day and night levels of hydroxyindole-O-methyltransferase activities in the latter animals were slightly less than the low daytime levels for the normal, sighted guinea pigs.


Acute and chronic effects of the fornix section on cyclic gonadotropin secretion and ovulation in the rat.

The fornix was sectioned in the frontal plane by means of a razor blade knife, and acute and chronic effects of this section on gonadotropin secretion were estimated. The 5-day cyclic rat which received the section of fornix under either anesthesia at 12:00 on the day of diestrus II showed advancement of the proestrous and estrous vaginal smears and as well as ovulatory gonadotropin release by one day. It was revealed that the primary effect was the inducement of FSH release on the day of section. The 4-day cyclic rat bearing the fornix section chronically resumed vaginal cyclicity after elapsing the diestrous period for 18 to 25 days. The rat ovulated normally and mean number of ova inoviducts was not different from that in the intact rat. However, the sectioned rat hadan higher concentrations of pituitary and serum FSH on the day of diestrus II than thatin the intact rat, and had an higher concentration of serum LH on the day of estrus. These results indicate that the hippocampus exerts the inhibitory influence on LH and FSH release and if this is eliminated the facilitatory influence dominates the brain mechanism controlling gonadotropin release, resulting in the advancement of estrous cycle (the acute effect) or the increase of gonadotropin release (the chronic effect).


Inhibition of ovulation in the rat by electrical stimulation of the lateral amygdala.

Effects of electrical stimulation of the lateral amygdaloid areas on ovulatory gonadotropin release were examined in adult Wistar female rats. Electrical stimulation was applied in rats in proestrus under ether anesthesia with square wave pulses of 0.5 ms duration and 100 Hz frequency for 30 min (30 sec on and off). Stimulation of the lateral amygdala blocked ovulation in 50% of animals when it was applied between 13:30 and 14:30 with a current of 300 muA. Stimulation of the medial amygdala under the same experimental condition was absolutely ineffective to block ovulation. Sham stimulation was also ineffective. In determining the gonadotropin concentration in serum, the stimulation into the lateral amygdala was observed to inhibit the ovulatory release of LH, FSH and prolactin. It may be said that the lateral amygdaloid area participates in the control of gonadotropin release in an inhibitory manner.


Tissue acetylsalicylic acid esterase activity in rats with acute and chronic liver damage from carbon-tetrachloride and ethanol.

The hydrolysis of acetylsalicylic acid (ASA) in vivo by serum and various tissues of rats with experimentally produced acute and chronic toxic liver damage, was estimated by spectrophotometric measurement of salicylic acid (SA) appearance. It was inferred from the data obtained that the liver tissue helped to maintain normal ASA esterase activity in the blood which would otherwise be affected by liver damage.


High-molecular-weight DNA polymerases from mouse myeloma. Purification and properties of three enzymes.

Cytoplasmic (high-molecular-weight) DNA polymerase was partially purified from mouse myeloma. Upon chromatography on DEAE-Sephadex, following fractionation on phosphocellulose, the enzyme was resolved into three species named CI, CII, and CIII. The species CI and CII have equal sedimentation coefficients (10.5 S) in sucrose gradients without salt. In the presence of 125 mM ammonium sulfate the sedimentation coefficients are reduced to 8.6 S. The species CIII shows sedimentation coefficients of 5.7 S and 5.2 S without salt and in the presence of 125 mM ammonium sulfate, respectively. This species is assumed to be an artifact arising from either CI or to a minor extent from CII. The optima for pH, KCl and Mg2+ concentration, and the extent of inhibition by N-ethylmaleimide are the same. However, the enzymes differ in their responses to Mn2+ (substituting for Mg-2+), and to addition of ethanol, dimethylsulfoxide, and various phospholipids in the assay mixture. The enzymes prefer poly[d(A-T - d(A-T)] or partially degraded (activated) DNA as template rather than double-stranded or single-stranded DNA. The activity on activated DNA relative to that on poly[d(A-T) - D(A-T)] was found to be 93, 66, and 29% for DNA polymerases CI, CII, and CIII, respectively.


Discontinuous DNA replication in mouse P-815 cells.

The length of newly synthesized DNA strands from mouse P-815 cells was analyzed after denaturation both by electrophoresis and by sedimentation in alkaline sucrose gradients. [3-H]-Thymidine pulses of 2-8 min at 37 degrees C predominantly label molecules of 20-60 S. With 30-s pulses at 25 degrees C, all the [3-H]thymidine appears in short DNA strands of 50-200 nucleotides. Thus, DNA strand elongation occurs discontinuously via Okazaki fragments at both the 5' end and the 3' end. In dodecylsulfate lysates, only 10% of the Okazaki fragments are found as single-stranded molecules. About 90% are resistant to hydrolysis by the single-strand-specific nuclease S-1 and band in isopycnic gradients at the buoyant density of double-stranded DNA. No evidence for ribonucleotides at the 5' end of Okazaki fragments was obtained either in isopycnic CsCl or Cs2SO4 gradients or after incubation with polynucleotide kinase and [gamma-32P]ATP.


A pulse fluorometry study of lipoamide dehydrogenase. Evidence for non-equivalent FAD centers.

The time dependence of the fluorescence of tryptophanyl and flavin residues in lipoamide dehydrogenase has been investigated with single-photon decay spectroscopy. When the two FAD molecules in the enzyme were directly excited the decay could only be analyzed in a sum of two exponentials with equal amplitudes. This phenomenon was observed at 4 degrees C (tau-1 = 0.8 ns, tau-2 = 4.7 ns) and at 20 degrees C (tau-1 = 0.8 ns, tau-2 = 3.4 ns) irrespective of the emission and excitation wavelengths. This result reveals a difference in the nature of the two FAD centers. By excitation at 290 nm the fluorescence decay curves of tryptophan and FAD were obtained. The decays are analyzed in terms of energy transfer from tryptophanyl to flavin residues. The results, which are in good agreement with those obtained previously with static fluorescence methods, show that one of the two tryptophanyl residues within the subunit transfers its excitation energy to the flavin located at a distance of 1.5 nm.


Clinico-pathological correlations in a case of primary angiosarcoma of the pericardium.

A case of angiosarcoma of the pericardium in a 30-yr-old man is reported. The clinical diagnosis of pericardial neoplasm was assumed on the basis of angiocardiography. The pathological findings of the heart are shown in detail. A survey of the clinico-pathological data in the 15 reported cases of angiosarcoma of the pericardium is presented.


Dopaminergic neurons in the nigro-striatal and mesolimbic pathways: mediation of specific effects of D-amphetamine.

The administration of d-amphetamine (15 mg/kg, i.p.) to rats causes stereotypy, hypothermia among animals placed in a cold environment, and paradoxical behavioral thermoregulation (i.e., animals in a cold environment choose not to place themselves under the beam emitted by a heat lamp). These effects are blocked in animals lesioned unilaterally in the mesolimbic dopaminergic projections to the olfactory tubercule and nucleus accumbens. In contrast, a unilateral lesion destroying the nigro-striatal projections within the caudate nucleus blocks none of these responses, and actually potentiates the induction of stereotypy by d-amphetamine. Both lesions cause the animal to exhibit rotational behavior in response  to the subsequent administration of d-amphetamine. These observations suggest that the mesolimbic dopaminergic projections mediate some of the behavioral and visceral effects of d-amphetamine.


A model for the rapid development of dispositional and functional tolerance to barbiturates.

The s.c. implantation of a 75 mg pentobarbital pellet in the back of a conscious mouse resulted in a much more rapid development of tolerance to barbiturates than that produced in mice receiving daily i.p. injections of 75 mg/kg sodium pentobarbital. Acceleration in tolerance development by pentobarbital pellet implantation was evidenced by a decrease in sleeping time after the challenge with either sodium pentobarbital or sodium barbital. The degree of hepatic microsomal drug enzyme induction after pentobarbital pellet implantation also was found to be significantly higher than that produced by the injection technique. Further studies demonstrated that the threshold for pentylenetetrazol-induced seizures was significantly reduced compared to that of the sodium pentobarbital daily injected and control groups. These studies provide an animal model for studying the mechanism of barbiturate tolerance and dependence.


On a prime role for newly synthesized dopamine in striatal function.

Rats were given either the tyrosine hydroxylase inhibitor, alpha-methyltyrosine (alphaMT), in doses of 10 or 250 mg/kg or the neuroleptic, haloperidol (0.25 mg/kg). Other rats received both drugs (haloperidol 30 min after alphaMT). This dose of haloperidol alone caused only a slight, gradually developing catalepsy, while alphaMT alone caused none. The combination quickly caused a strong catalepsy. Striatal dopamine (DA) stores were only minimally depleted at the time of catalepsy potentiation. Th e marked elevation of striatal homovanilluc acid concentration seen after haloperidol administration was greatly inhibited by alphaMT pretreatment. It is concluded that the marked potentiation of haloperidol-induced catalepsy by alpha MT is related to the absence of newly synthesized DA rather than to an exhausted main DA pool and that newly synthesized DA has a greater role in striatal function than does DA of the main striatal storage pool.


Automutilation induced by clonidine in mice.

Clonidine (2-(2,6-dichlorophenylamino)-2-imidazoline hydrochloride, St155, Catapres), after administration of a single large dose, was found to induce automutilation in mice housed individually in the absence of objects to bite. This abnormal behavior was not significantly altered with chronic administration of the drug, and showed no behavior induced by the same drug in mice caged in groups.


Differences in the nature of the stereotyped behaviour induced by aporphine derivatives in the rat and in their actions in extrapyramidal and mesolimbic brain areas.

Apomorphine, (minus)-N-n-propylnorapomorphine [ (minus)-NPA ] and (plus or minus)-N-n-propylnorapomorphine [ (plus or minus)-NPA ] each caused stereotyped behaviour patterns in the rat which could be differentiated into two components, sniffing and repetitive head and limb movements (low intensity component) and gnawing, biting and licking (high intensity component). Low intensity components occurred at low doses of apomorphine and high intensity components at larger doses but the two components never occurred independently for (minus)-NPA or (plus or minus) NPA. Biting was the predominant effect of these agents which were shown to be at least twenty times more potent than apomorphine. The (minus)-isomer of NPA was the more potent. The two components of stereotypy were differentiated both pharmacologically (using amantadine, reserpine plus alpha-methyl-p-tyrosine and haloperidol) and by lesions placed in areas of the extrapyramidal (caudate--putamen, globus pallidus, substantia nigra) and mesolimbic (nucleus accumbens septi, tuberculum olfactorium, nucleus amygdaloideus centralis) systems. However, both sniffing and biting responses  were reduced by lesions of the serotonergic raphe nuclei. The two stereotypic components were differentially induced by intracerebral injections of apomorphine and (minus)-NPA into the caudate--putamen, nucleus accumbens septi and tuberculum olfactorium. Injections into the central nucleus of the amygdala were ineffective. The degree of involvement of the different areas was shown to differ for apomorphine and (minus)-NPA, in particular the nucleus accumbens septi appeared more important for the action of (minus)-NPA and the tuberculum olfactorium for apomorphine. Intracaudate (minus)-NPA was less active than apomorphine but, generally, intracerebrally applied (minus)-NPA was twice as potent as apomorphine. Both (minus)-NPA and apomorphine caused circling behaviour in animals with asymmetric medial raphe nucleus lesions (contralateral) or unilateral lesions of the substantia nigra (ipsilateral). In these experiments (minus)-NPA was ten times more potent than apomorphine.


Catecholamine correlates of isolation-induced aggression in mice.

Drugs differentially affecting catecholaminergic neurotransmission mechanisms were injected into isolated mice to assess effects on aggression. L-DOPA in combination with a peripheral decarboxylase inhibitor produced a dose-dependent decrease in the number of fights and average fight durations accompanied by an increase in attack latencies. Central dopamine levels were increased whilst brain noradrenaline and 5-hydroxytryptamine were reduced. Virtually identical effects on aggression were observed after apomorphine. Pimozide decreased fighting, but neither attack latencies nor average fight durations were effected. All doses of disulfiram virtually abolished all components of aggressive responses. The effect of D-amphetamine on number of fights was biphasic; lower doses potentiated aggression, but higher doses reduced fighting accompanied by increased attack latencies and reduced average fight durations. We conclude that catecholamines may be intimately involved in mediating certain components of aggressive responses but that balance relationships among various putative neurotransmitters may be a more meaningful correlate of this behavior.


Acute oxygen supply by infusion of hemoglobin solutions.

Preparations of 6% human hemoglobin have been investigated for 4 years. While the oxygen dissociation curve is shifted to the left, the hemoglobin solution has been shown to deliver oxygen adequately to the tissues in animal studies. Intracorporeal fate has been investigated by 131-I tagging, and the hemoglobin has not been found to accumulate in the tissues. Intravascular half-life has varied between 1.5 and 3.5 hours.


Prostaglandin F2alpha: modification of its abortifacient effect by depo-estradiol cypionate in rats.

Insertion of a Silastic-PVP implant containing 150 mug PGF2alpha into one of the uterine horns or administration of a single subcutaneous injection of 4 mug of Depo-Estradiol Cypionate (ECP) did not induce abortion in pregnant rats. However, a combined treatment with 150 mug of PGF2alpha in a Silastic-PVP implant and 4 mug of ECP induced abortion in all treated rats. Apparently, ECP stimulated endogenous production of PGF and consequently enhanced the effect of the administered 150 mug of PGF2alpha. The release of 3H-PGF2alpha from Silastic-PVP implants was faster after intrauterine or intraperitoneal than after subcutaneous insertion. The release rate of 3H-PGF2alpha from Silastic-PVP implants in vitro was comparable to that observed after subcutaneous placement of similar implants. It is suggested that administration of estrogen combined with PGF2alpha might shorten the interval between treatment with PGF2alpha and abortion. Moreover, a Silastic-PVP implant would lend itself as a better method of administration, releasing PGF2alpha locally, slowly, and continuously.


The use of a radioreceptorassay of human chorionic gonadotropin for the diagnosis and management of ectopic pregnancy.

The radioreceptorassay of human chorionic gonadotropin (hCG), with a sensitivity of 50 pg or 3 mIU/ml of plasma, has provided almost 100% reliability in detecting pregnancy after the first missed cycle. This test may be performed within 1 hour and is ideally suited to the clinical detection of ectopic pregnancy, especially in patients who require immediate surgical intervention. Thirteen patients with suspected ectopic pregnancy were evaluated by the radioreceptorassay, one of whom was followed with four separate determinations. The results of the assay were subsequently compared with those of hemagglutination pregnancy tests, clinical symptoms, and pathologic findings. All of the patients were diagnosed accurately by the radioreceptorassay, even when hemagglutination tests yielded a false indication of pregnancy. By this assay, the hCG levels during ectopic pregnancies are generally lower than those found during a normal intrauterine pregnancy; in addition, pregnancy may be detected much earlier (prior to the rupture) than is possible by hemagglutination tests. Furthermore, the diagnosis of ectopic pregnancy may be excluded for patients admitted to the hospital with acute abdominal emergencies.


Physiologic and pharmacologic studies on the motility of isolated guinea pig ovaries.

The spontaneous motility of ovaries isolated from guinea pigs in early and late proestrus and estrus was explored. The influences of oxytocin, prostaglandin  F2alpha (PGF2alpha), and adrenergic agonists and antagonists were also studied. Spontaneous ovarian isometric developed tension (IDT) and rate of contractions (RC) were greater in late proestrus than in early proestrus or estrus. Furthermore, in late proestrus, oxytocin and PGF2alpha induced ovarian motilities of comparable magnitude and significantly greater than those elicited in other stages of the cycle. Norepinephrine and phenylephrine either failed to alter or inhibited IDT and RC of ovaries in estrus, but stimulated motility in the presence of propranolol. The depressive influence of norephinephrine upon the IDT of ovaries in early proestrus was not seen in late proestrus, when the neurotransmitter was clearly stimulating. The hormonal status of guinea pigs seems to influence spontaneous ovarian motility as well as pharmacologic reactivity to several agents, including those presumably acting upon alpha- and beta-adrenergic receptors.


Cumuli oophori in tissue culture: hormone production, ultrastructure, and morphometry of early luteinization.

The steroidogenic activity of cumulus oophorus granulosa cells obtained from unstimulated rabbits in estrus has been investigated at closely timed intervals in short-term cultures. The presence of viable, structurally well preserved oocytes and the addition of large follicle follicular fluid had no apparent inhibitory effect on progesterone or estrogen synthesis and secretion. Progesterone secretion was especially stimulated by the addition of follicle-stimulating hormone (FSH) and luteinizing hormone (LH) to the culture medium. A similar increment was also seen, although inconsistently, for estrogen secretion. Ultrastructural evidence of luteinization was demonstrated by quantitative morphometric techniques in control cultures as early as 6 hours after explantation and was shown to relate well to biochemical luteinization. It is suggested that the main modifications in cytoplasmic organelles concomitant with luteinization may be represented only by volume and surface changes rather than by alterations in morphology. Therefore, such changes need to be assessed quantitatively. Oocytes exhibited isolated maturative changes almost exclusively in FSH-LH cultures, such as cortical granule synthesis, resumption of meiosis, polar body extrusion, and two-cell parthenogenetic cleavage. It is suggested that cumulus granulosa cells may play a significant role in the resumption of "spontaneous" maturation in oocytes after their release in vitro from their follicles. Characteristic, thin-walled vesicles, either isolated or in rosette-like formation with cumulus granulosa cells, were described. A possible common nature was suggested for these vesicles and the so-called "Call-Exner bodies."


Testosterone rebound therapy in the treatment of male infertility.

Large doses of testosterone were used to cause degeneration of the testicular germinal epithelium and a subsequent rebound of spermatogenesis, to produce improved seminal fluid. A 29% pregnancy rate was obtained by 131 patients treated for idiopathic oligospermia, and an 8% pregnancy rate was obtained by 12 patients treated for azoospermia. The seminal fluid of three patients (2%) was permanently damaged by the therapy.


The effect of maternal immunity, age at vaccination and doses or live vaccines on immune response to Newcastle disease.

The short life span of domestic fowls necessitates the use of early vaccination, but the response is affected by the immaturity of the immune mechanism and by the presence ofmaternal immunity: this results in an uneven immunity of the large and continually changing poultry population. This paper compares the response of groups of chicks from vaccinated and non-vaccinated hens after vaccination at various ages with Newcastle Disease vaccines of different pathogenicity (lentogenic and mesogenic strains). The results show that: 1. maternal immunity has a great effect on the pathogenicity of the vaccine viruses and the subsequent immune response; 2. the older the chicks are at the time of vaccination, the better is their immune response; 3. for vaccines of low pathogenicity the immune response can be enhanced by increasing the dose of the virus irrespective of the presence of maternal immunity.


Vaccination against bovine respiratory disease.

Vaccination is but one element in a control programme for bovine respiratory disease. Its laboratory study can be divorced from the others but its field application cannot. The problems associated with the development of effective vaccines fall into two broad groups: multiplicity and ubiquity of pathogens and secondly the identification of the crucial elements in an immune response. Agricultural systems which experience annual outbreaks of respiratory disease attributable to the same pathogen in cattle of specific age have the choice of using passive or active immunity of minimal valency. In the majority of systems the cause and timing of an outbreak cannot be predicted and therefore multivalent vaccines are required. Both inactivated and modified live products are available for use against the well-known pathogens. Their relative advantages hinge on the significance attributed to the ability to stimulate the production of particular immunoglobulins at specific body sites and the persistence of the responses. The widely held view that success requires the stimulation of secretory antibodies by intranasal administration of living vaccines is not universally accepted. An assessment of their protective value is not easily made because of the difficulty of reproducing an adequate field challenge in the laboratory. The measurement of serological responses and virus shedding times following challenge are of limited value as alternatives.


Culture of Zollinger-Ellison tumor cells.

We have successfully grown six Zollinger-Ellison tumors in vitro with use of a monlayer tissue culture technique. The initial gastrin concentration in the medium varied between 0 and 100 ng per ml. Many cytoplasmic secretory granules were seen in the cells of one culture population. Gastrin secretion was stimulated by the addition of fresh medium to the culture flasks. Both the culture cells and the medium were found to contain primarily big gastrin (G-34) but smaller amounts of little gastrin (G-17) were also present. Gastrin concentration in the medium decreased with time in culture until no hormone was detected between 2 and 6 weeks, possibly because of endocrine cell dedifferentiation and an increased proportion of fibroblasts in the population.


[Diagnosis of an ectopic pregnancy by means  of ultrasonic-scan-technique].

A case of an ectopic pregnancy is reported, which was diagnosed by ultrasonic-scan-technique. The echogramm had shown beside an enlarged uterus on the left side of the patient, a cystic structure on the right side; inside this cystic a fetus with spontaneous movements could be observed. The immediate following surgical intervention verified the ultrasonic diagnosis, the fetus showed spontaneous as well as by touching induced movements up to 30 minutes.


The effect of cholestyramine on intestinal absorption.

Cholestyramine in a mean dosage of 0-6 g/kg/day has been given to 18 children with familial hypercholesterolaemia for between one and two and a half years. With prolonged treatment folate deficiency occurred, as evidenced by a fall in the mean serum folate concentration from 7-7 ng/ml before treatment to 4-4 ng/ml for patients on treatment for over one year; a corresponding lowering of red cell folate was also seen. Oral folic acid 5 mg daily overcame this depletion, and should be given to all patients on long-term anion exchange resins. Prothrombin time has remained normal in all patients; there has been a significant decrease in the mean serum concentrations of vitamins A and E and of inorganic phosphorus over the first two years of treatment, although values remain within the normal range. The routine administration of fat-soluble vitamins appears unnecessary but it is prudent to measure prothrombin time and serum vitamins A and E at intervals. In children who were having a normal intake of dietary fat five out of seven tested had faecal fat of over 5 g/day while on cholestyramine. No chold has developed diarrhoea, and growth has been normal. The concentrations of serum iron, vitamin B12, plasma calcium, and protein did not change significantly in any patient.


Transport across the rabbit foetal yolk-sac of fractions of IgG from several mammalian species.

Electrophoretically migrating fast and slow IgG fractions of rabbit, mouse, human, guinea-pig, dog, horse and cow sera were prepared by DEAE-cellulose chromatography, labelled with either 131I or 125I, and tested in passage across yolk-sac splanchnopleur of rabbits. In foetal sera, slow IgG generally reached higher concentrations than fast IgG, but even with mouse Ig, which has a high ratio between the two fractions, the difference was only four-fold. This differential between fast and slow IgG was not sufficient to explain the absorptive selectivity between IgG of different species. The capacity of isotopically labelled rabbit and equine IgG to be transported across the rabbit foetal yolk sac was also studied using fractions prepared by isoelectric focusing. Marked differences in concentration quotient (CQ) values (CQ equals (concentration in foetal serum)/(concentration in injected material)) between individual pI peaks for these species were found.


Shark attack in Natal.

The injuries in 5 cases of shark attack in Natal during 1973-74 are reviewed. Experience in shark attacks in South Africa during this period is discussed (1965-73), and the value of protecting heavily utilized beaches in Natal with nets is assessed. The surgical applications of elasmobranch research at the Oceanographic Research Institute (Durban) and at the Headquarters of the Natal Anti-Shark Measures Board (Umhlanga Rocks) are described. Modern trends in the training of surf life-guards, the provision of basic equipment for primary resuscitation of casualties on the beaches, and the policy of general and local care of these patients in Natal are discussed.


Cytotoxic effect of lymphocytes from pregnant mice on cultivated tumor cells. I. Specificity, nature of effector cells and blocking by serum.

Lymph-node and spleen cells from pregnant BALB/c mice were found to be cytotoxic (by a microcytotoxicity assay) to cultivated syngeneic tumor cells. Target cells included a series of in vitro transformants derived from BALB/c 3T3 cells (3T3-MSV,3T3-SV40 and 3T12), as well as two lines of tumors induced in vivo. Skin fibroblasts and non-transformed 3T3 cells were not similarily affected. The cytotoxic lymphocyte effect was decreased by treatment with anti 0 serum and complement but was not removed by passage through a column removing cells with immunoglobulin at their surfact. It could be blocked by serum taken from pregnant mice and from mice carrying methylcholanthrene-induced sarcomas. These sarcomas could be different from the ones used as targets in vitro, but the blocking effect was higher when serum was tested from mice carrying the respective tumor studied. The blocking activity of serum from pregnant mice was removed by absorption with neoplastic cells, taken either from culture or directly from mice, and with fresh embryonic cells, but not to the same extent by absorption with explained fibroblasts, 3T3 cells, or adult kidney cells. Absorption was most effective when the same line of cells was used as targets. Both the cytotoxic lymphocyte effect and the blocking serum activity were seen already during the first pregnancy and lasted for at least 1 month subsequent to delivery. Serum from pregnant mice could "arm" control lymph-node cells and produce a lymphocyte-dependent cytotoxic effect on neoplastic, but not on control, target cells.


Cytotoxic effect of lymphocytes from pregnant mice on cultivated tumor cells. II. Blocking and unblocking of cytotoxicity.

Sera from BALB/c mice transplanted with either of two syngeneic, methylcholanthrene-induced sarcomas (1315 and 1321) were found to block (i.e. abrogate) the cytotoxic effect of lymph-node cells (LNC) from multiparous mice on cultivated tumor cells. The blocking activity was seen even at high serum dilutions (up to 1:320). It was found to persist in mice whose tumors had been removed 8-11 days previously. These mice had transplantation resistance against individually unique antigens of the respective sarcoma to which they had been immunized, but not against any common, putatively embryonic ones. In keeping with this finding, their sera did not block LNC-mediated cytotoxicity to the unique transplantation antigens, when tested 8-11 days after removal of the immunizing tumor. Sera from rabbits immunized with BALB/c embryonic tissues and subsequently absorbed with normal adult BALB/c cells, were found to cancel ("unblock") the blocking activity of serum from multiparous mice. The specificity of this unblocking effect and its potential usefulness as a tool to study immune reactions to embryonic tumor antigens need to be established.


Effect of testosterone and estradiol-17beta on synthesis of DNA, RNA and protein in human breast in organ culture.

Effects of testosterone (T) and estradiol-17beta (E-2) on the synthesis of DNA, RNA and protein were studied in explants of the following types of malignant and non-malignant human female breast grown in organ culture: cystic mastitis (7 cases), fibroadenoma (8), carcinoma (17) and uninvolved tissue from cancer-bearing breast (8). In cultures of systic mastitis T uniformly inhibited the incorporation of thymidine-3H into DNA, uridine-3H into RNA and L-amino acids-14C (AA-14C) into protein. E-2 inhibited the incorporation of thymidine-3H, but had a variable effect on the incorporation of uridine-3H and AA-14C. Cultures of fibroadenoma, with no steroid added, were highly proliferative especially at the outer surface, but the incorporation of thymidine-3H, uridine-3H and AA-14C was much lower than in cystic mastitis. Whereas in fibroadenoma T uniformly inhibited the synthesis of DNA, RNA and protein, the effect of E-2 was variable: in most cases it inhibited the synthesis of DNA,but in one it stimulated it appreciably; in the majority of cases E-2 stimulated RNA and protein synthesis. In cultures of cancerous tissue T depressed, in most cases, the incorporation of thymidine-3H, uridine-3H and AA-14C, but it stimulated it in 4 out of 17. E-2 inhibited the synthesis of DNA, RNA and protein in 6 cases (2 of them were inhibited by T), stimulated them in 9 (one stimulated by T) and had no clear effect in 2. The effect of the steroids on the explants of uninvolved tissue was variable and did not always parallel their effect on the cancerous tissue from the respective patients.


A permeability test for the study of mitochondrial injury in in vitro cultured heart muscle and endothelioid cells.

A cytochemical permeability test for the detection of injury to in situ mitochondria of cultured heart cells is presented. The test is based on the increased rate at which injured mitochondria stain for succinate dehydrogenase activity. Whereas an intact inner mitochondrial membrane limits the rate at which Nitro Blue tetrazolium and phenazine methosulphate reach succinate dehydrogenase, injured mitochondria allow these reactants to reach the enzyme more rapidly to form microscopically-observable formazan granules. The extent of staining at fixed durations of incubation with the reactants was assessed on a blind basis with pseudo dark-field microscopy, using a standardized rating scale. Differences in the staining of control and treated cells were analysed statistically by a semi-quantitative method. Treatment of the cultures with either vitamin A or chlorpromazine, resulted in more rapid mitochondrial staining. Brief pre-fixation of the cells with cold acetone also labilized the mitochondria as did a delay in the change of culture medium.


Combined staining of protein-bound sulphydryl groups and DNA in polyacrylamide model systems.

A model system of polyacrylamide films containing protein and DNA has been used to examine the feasibility of combining the dihydroxydinaphthyldisulphide (DDD)-diazonium salt procedure for localizing protein-bound sulphydryl groups with the Feulgen technique for DNA to make possible the direct measurement of both these parameters simultaneously. Optimun conditions for the sulphydryl group reaction require reduction of the protein-containing films in 10% aqueous ammonium sulphide for 3 hr at 50 degree C followed by treatment with a DDD solution at 50 degree C for 4 hr. The final coloured product was developed in a solution of the diazonium salt, Fast Red TR, for 15 min. The azo compound thus produced was completely resistant to hydrochloric acid hydrolysis in the manner of the Feulgen reaction. Calculation of protein-bound sulphydryl groups and DNA from measurements made on doubly-stained films showed excellent agreement between the measured and the expected values.


Utilization of L-cell nucleoside triphosphates by Chlamydia psittaci for ribonucleic acid synthesis.

Long-term, 32-P-labeled L cells were infected with the obligately intracellular parasite Chlamydia psittaci (strain 6 BC). At 20 h postinfection, [3-H]uridine was added, and the infected cells were sampled at intervals for incorporation of the labels into the uridine triphosphate (UTP) and cytidine triphosphate (CTP) pools of the host L cell and the uridine monophosphate (UMP) and cytidine monophosphate (CMP) in 16S ribosomal ribonucleic acid (RNA) of the parasite. The specific activity of the nucleotides was calculated from the ratio of 3-H to 32-P counts in the nucleotides. The rate of approach to equilibrium labeling of UTP and CTP in L-cell pools and UMP and CMP in 16S RNA from the exogenous uridine label was determined from the increase in the ratios of the specific activities of CTP to UTP and CMP to UMP with time. The rate of approach to equilibrium CMP:UMP labeling of the 16S RNA of C. psittaci was consistent with the rate predicted from the kinetics of labeling of the CTP and UTP pools of the host L cell. In analogous experiments, the rate of approach to equilibrium guanosine monophosphate:adenosine monophosphate labeling of 16S RNA from an exogenous [14-C]adenine label was consistent with the rate predicted from the kinetics of labeling of the purine nucleoside triphosphate pool of the host cell. These results support the concept that members of the genus Chlamydia owe their obligate intracellular mode of reproduction to a requirement for energy intermediates which is fulfilled by the host cell. In addition, evidence was obtained that the total acid-soluble purine nucleoside triphosphate pool of L cells accurately represents the precursors of L-cell 18S ribosomal RNA.


Structural similarity of the membrane envelopes of rhizobial bacteroids and the host plasma membrane as revealed by freeze-fracturing.

The freeze-fracture technique was used to study the host plasma membrane and the membrane envelope of bacteroids in rhizobial root nodules of three host-rhizobium combinations. In all three combinations studied, the membrane envelopes of bacteroids are structurally similar to their host plasma membrane. However, the membrane appears to be reversed, because the number and arrangement of particles in the outer fractured face (face A, concave) and in the inner fractured face (face B, convex) of the host plasma membrane are seen, respectively, in the inner fractured face (face B, convex) and in the outer fractured face (face A, concave) of the membrane envelope of the bacteroids at an early stage. This reversion of the membrane surface is consistent with the hypothesis that the membrane envelopes of bacteroids are derived from the host plasma membrane during endocytotic engulfment.


Plasmalogen composition of Anaeroplasma.

The polar lipids of Anaeroplasma contained 33.1 percent alk-1'-enyl glyceryl ether (plasmalogen) form. Phosphatidylglycerol was the major polar lipid (55.2 percent) and contained nearly all of the plasmalogen. The alk-1'-enyl glyceryl ether form accounted for 58.3 percent of the phosphatidylglycerol.