Spore membrane(s) as the site of damage within heated Clostridium perfringens spores.

Clostridium perfringens spores were injured by ultrahigh-temperature treatment at 105 C for 5 min. Injury was manifested as an increased sensitivity to polymyxin and neomycin. Since many of the survivors could not germinate normally the ultrahigh-temperature-treated spores were sensitized to and germinated by lysozyme. Polymyxin reportedly acts upon the cell membrane. Neomycin may inhibit protein synthesis and has surface-active properties. Injured spores were increasingly sensitive to known surface-active agents, sodium lauryl sulfate, sodium deoxycholate, and Roccal, a quaternary ammonium compound. Injured spores sensitive to polymyxin and neomycin also were osmotically fragile and died during outgrowth in a liquid medium unless the medium was supplemented with 20% sucrose, 10% dextran, or 10% polyvinylpyrrolidone. The results suggested that a spore structure destined to become cell membrane or cell wall was the site of injury. Repair of injury during outgrowth in the presence of protein, deoxyribonucleic acid, ribonucleic acid and cell wall synthesis inhibitors was consistent with this hypothesis.


Synthesis of deoxyribonucleic acid, ribonucleic acid, and protein during sporulation of Clostridium perfringens.

The kinetics of deoxyribonucleic acid (DNA), ribonucleic acid (RNA), and protein synthesis as well as protein breakdown during sporulation by Clostridium perfringens were determined. Maximum levels of DNA and net RNA synthesis occurred 3 and 2 h, respectively, after inoculation of sporulation medium. The rate of RNA synthesis decreased as sporulation progressed. Deoxyadenosine increased uptake of [14C]uracil and [14C]thymine but depressed the level of sporulation and the formation of heat-resistant spores when added at concentrations above 100 mug/ml. Unlike Bacillus species, net protein synthesis, which was sensitive to chloramphenicol inhibition, continued during sporulation. The rate of protein breakdown during vegetative growth was 1%/h. During sporulation this rate increased to 4.7%/h. When added to sporulation medium at 0 time chloramphenicol reduced protein breakdown to 1%/h. If added at 3 h the rate decreased to 2.1%/h. The role of proteases in this process is discussed.


Properties of adenyl cyclase and cyclic adenosine 3',5'-monophosphate receptor protein-deficient mutants of Escherichia coli.

Several spontaneous cya and crp mutants of Escherichia coli have been selected as clones simultaneously resistant to phage lambda and nalidixic acid and characterized. Both cya and crp mutants have been found to grow as cocci with increased doubling times. They have increased resistance to some mutagens (methylmethanesulfonate, ultraviolet light, gamma rays), antibiotics (nalidixic acid, ampicillin), phages (lambda, T6), sublethal heat and hypotonic shock, and decreased resistance to neutral detergents (sodium dodecyl sulfate, sodium deoxycholate), a protein synthesis inhibitor (streptomycin), and a respiratory inhibitor (sodium azide). The nature of changes in cell parameters indicate fundamental alterations in the envelope structure of the cya and crp mutant cells. The new cya and crp mutants have been found to be multiply carbohydrate negative and nonmotile in conformity with similar previously isolated mutants. Studies of revertants and phi80 cya+ and phi80 cya transductants indicated that the pleiotropic phenotype is related to a single mutational event at the cya or the crp locus in the mutants.


Suppressor-induced structural changes in a missense L-ribulokinase of Escherichia coli.

A suppressor mutation specific for a missense codon in the L-ribulokinase structural gene of the L-arabinose operon of Escherichia coli B/r enhanced L-arabinose utilization by the strain containing the missense codon. Electrophoretic comparisons of the wild-type, missense, and suppressed missense L-ribulokinases indicated that the suppressor changed the structure of the missense kinase, thereby increasing its catalytic activity. Hyperinducibility imposed on an operator-distal gene by the missense codon was not affected by the suppressor mutation.


Lethal effect of mitomycin C on Haemophilus influenzae.

The sensitivity of ultraviolet-sensitive strains to inactivation by mitomycin C (MC) is at the most only a factor of two greater than that of the wild type. The presence of inducible prophage has very little effect on the sensitivity. Genes which control excision of ultraviolet-induced pyrimidine dimers also control repair of MC-induced cross-links, as measured by resistance of denatured deoxyribonucleic acid (DNA) from treated cells to S1 nuclease digestion. However, endonucleolytic breaks in MC-damaged DNA, as judged by decreased single-strand molecular weight upon incubation of treated cells, are independent of these genes and probably are caused by monoadducts. After long periods of incubation there is a return to the molecular weight of untreated DNA. DNA degradation after MC treatment of various strains is not correlated with sensitivity to inactivation. Stationary-phase cells of all strains are more than twice as sensitive to MC as exponentially growing cells, and the sensitivity difference agrees with the measured difference in the number of cross-links after MC treatment of cells in the two growth stages. Evidence has been obtained that these phenomena result from differences in uptake of MC, which can be influenced by cyclic adenosine monophosphate. Small deviations in MC sensitivity from that of the wild type observed in mutants lacking the adenosine 5'-triphosphate-dependent nuclease are postulated to result from differences in MC uptake. These mutants, although no more ultraviolet sensitive than the wild type, are more sensitive to streptomycin, which also must be taken up by the cell to be effective.


Galactosaminogalactan from cell walls of Aspergillus niger.

A new heteropolysaccharide has been isolated by alkaline extraction of hyphal walls of Aspergillus niger NRRL 326 grown in surface culture. Its composition by weight, as determined by paper and gas chromatography and colorimetric analyses, is 70% galactose, 20% galactosamine, 6% glucose, and 1% acetyl. Two independent experiments have been used to ascertain copolymer structure: permeation chromatography in 6 M guanidinium hydrochloride, with controlled-pore glass columns of two fractionation ranges, and nitrous acid deaminative cleavage of galactosaminogalactan followed by reduction of fragments with [3H]borohydride and gel filtration chromatography. One of the tritiated fragments is tentatively identified as the disaccharide derivative galactopyranosyl 2,5-anhydrotalitol, on the basis of chromatographic properties and by kinetics of its acid hydrolysis. Smith degradation, methylation, deamination, and optical rotation studies indicate that the galactosaminogalactan consists of a linear array of hexopyranosyl units joined almost exclusively by alpha-(1 leads to 4) linkages. Hexosaminyl moieties are distributed randomly along the chains, which have an average degree of polymerization of about 100. The possible significance of this macromolecule in hyphal structure is considered.


Transformation of Escherichia coli by chromosomal deoxyribonucleic acid from Salmonella typhi.

The genetic transformation of Escherichia coli K-12 with chromosomal deoxyribonucleic acid purified from Salmonella typhi is described.


Studies on cytochrome oxidase. Preliminary characterization of an enzyme containing only four subunits.

The preparation of a four-subunit enzyme from yeast, capable of catalyzing the oxidation of ferrocytochrome c, is described. It is derived from proteins containing seven or five subunits by means of recycling exclusion chromatography in the presence of 0.1% sodium dodecyl sulfate. Its catalytic properties are similar to those of the parent enzyme. Gel electrophoresis of this preparation in the presence of sodium dodecyl sulfate reveals three bands, migrating with RF values that correspond to molecular weights of 14.6, 12.3, and 10.6 X 10(3), with the largest exhibiting an apparent 2:1 stoichiometry relative to the other two. Visible spectra in the region of 390 to 630 nm do not show any detectable difference from that of the parent cytochrome oxidase, while its heme a and copper content are raised to values around 20 nmol or ng atoms/mg of protein, respectively, corresponding to minimal molecular weights of 50 X 10(3). The molecular weight determined by physical means equals 107 X 10(3). Thus the enzyme probably contains two copies of each subunit. After extensive dialysis to remove as much as possible of the sodium dodecyl sulfate used in its preparation, this enzyme remains in solution in phosphate buffer in the absence of any added detergent, while under similar conditions the seven-subunit complex precipitates completely. A similar preparation can also be obtained from beef heart. The significance of these findings is discussed with respect to the role of the large subunits in the function as well as the biogenesis of the mitochondrial cytochrome oxidase complex.


Interaction of glucocorticoid receptor-steroid complexes with acceptor sites.

The binding of the "activated" receptor-glucocorticoid complexes of cultured rat hepatoma cells to nuclei, chromatin, and DNA has been studied under cell-free conditions. A critical factor in determining the shape of the binding curve is shown to be an inhibitory material which is present in crude cytosol and which can be removed without destroying the receptor-steroid complex. These and other results argue that the apparent saturation observed in earlier experiments may have been due to the inhibitors. Thus, the actual number of acceptor sites in hepatoma tissue culture cell nuclei is much larger than previously estimated and their affinity for the complex is lower. Nuclear binding experiments indicate that the inhibitory material interacts with the receptor-steroid complex. The inhibitors appear to be macromolecular; but their effects cannot be mimicked by albumin or hemoglobin. The acceptor capacity at low ionic strength for binding receptor-glucocorticoid complexes increases when proceeding from nuclei to DNA. An analysis of the kinetics of association and dissociation and of the relative binding behavior of nuclei and DNA argues that the affinity of complex for nuclei is much greater than for DNA. DNA-associated histones reduce the amount of complex that binds to DNA. These and perhaps other chromosomal proteins may be responsible for the ordering of acceptor capacity. Evidence is presented that the difference in affinities of nuclear and DNA acceptors could also be due to chromosomal proteins. In nuclei, these proteins may thus both reduce the amount of complex binding by rendering regions of DNA less accessible and increase the binding affinity of some, or all, of those DNA binding sites which remain exposed.


Group translocation of the ribose moiety of inosine by vesicles of plasma membrane from T(3 cells transformed by Simian virus 40.

Plasma membrane vesicles are isolated from Simian virus 40-transformed Balb/c mouse 3T3 (SV-3T3) cells. These membrane vesicles contain no significant contamination by mitochondria, endoplasmic reticulum, or lysosomes as determined by marker enzyme analysis. The use of [U-14C] inosine as a transport substrate results in the accumulation of labeled ribose-1P as transport product by the plasma membrane vesicles. This suggests the action of purine nucleoside phosphorylase (the enzyme which mediates the phosphorolysis of inosine to ribose-1-P and hypoxanthine0 before, during, or after the transport step. Neither inosine nor significant amounts of hypoxanthine are found intravesicularly. The Km for inosine, the substrate in this reaction which leads to the accumulation of ribose-1-P by the plasma membrane vesicles, is 35 to 45 muM while the Vmax for ribose-1-P accumulation is 100 to 120 pmol/min/mg of plasma membrane protein...


Effect of L-3,4-dehydroproline on collagen synthesis and prolyl hydroxylase activity in mammalian cell cultures.

The incorporation of DL-3,4-dehydro[14C]proline into collagen and total protein of 3T3 cells occurred at approximately one-fifth the rate observed for L-[14C]proline. Addition of L-3,4-dehydroproline to the culture medium inhibited markedly the incorporation of [14C]glycine and L-[3H]lysine into the collagen of 3T3 cells, but there was only slight inhibition of the incorporation of the radiolabeled amino acids into total cellular proteins, indicating that the action of L-3,4-dehydroproline is specific for collagen. When 1 mM L-3,4-dehydroproline was added to the culture medium the [14C]hydroxyproline content was reduced 40% in the cell layer and 70% in the medium. The D isomer of 3,4-dehydroproline did not inhibit [14C]hydroxyproline formation. These findings indicate that L-3,4-dehydroline reduced the hydroxylation of the susceptible prolyl residues in the collagen molecule and the secretion of collagen from the cell. The reduction in the hydroxyproline content is probably related in part to a reduction in the activity of prolyl hydroxylase; when various mammalian cell cultures were exposed to 0.2 mM L-3,4-dehydroproline, the specific activity of prolyl hydroxylase was reduced markedly, while that of lysyl hydroproline, the specific activity of prolyl hydroxylase was reduced markedly, while that of lysyl hydroxylase was not affected. Under these conditions, cell growth and lactic dehydrogenase required protein synthesis. Removal of L-3,4-dehydroproline from  the growth medium resulted in a time-dependent increase in the specific activity of prolyl hydroxylase.


Effect of estrogen on gene expression in the chick oviduct. Correlation between nuclear-bound estrogen receptor and chromatin initiation site for transcription.

The [3H]estradiol exchange assay was used to characterize the nuclear estrogen receptor from the chick oviduct. After diethylstilibestrol (DES) treatment (14 days), the oviduct nuclei contained estrogen receptors that manifested high affinity (Kd = 2 X 10(-9)M) and low capacity binding (4000 to 5000 sites/cell) for estradiol. DES and estradiol competed significantly for [3H]estradiol binding, while testosterone and progesterone were ineffective. These binding sites were found in the oviduct and liver but not in the spleen, kidney, or muscle. Following salt extraction from nuclei, the receptor had a sedimentation coefficient of 4 S when analyzed by centrifugation in high and low salt sucrose density gradients. The [3H]estradiol exchange assay was used to examine the relationships between nuclear-bound receptor and RNA polymerase initiation sites on oviduct chromatin. Within 20 min after a single injection of 2.5 mg of DES to withdrawn chicks, a maximum number of estradiol receptor-binding sites was detected in oviduct nuclei. Within 30 min after DES injection, the total number of RNA initiation sites also increased, reaching 100% of control values. Daily injections of DES to unstimulated chicks resulted in a gradual increase in the nuclear content of estradiol receptor, which reached a maximum at 6 days and thereafter declined gradually up to 18 days of hormone treatment. A maximum number of initiation sites for RNA synthesis was also attained at 4 to 6 days of DES treatment and thereafter declined. When DES was withdrawn after 14 to 18 days of hormone stimulation, the numbers of nuclear estradiol receptor sites and initiation sites for RNA synthesis both declined gradually, reaching half-maximal values in 3 days and approached control values after 7 to 8 days of withdrawal. The increase in the concentration of nuclear estradiol receptor sites and the number of initiation sites for RNA synthesis also showed a close correlation with the dosage of DES administration. Both attained maximum levels at 1.25 mg of DES with a half-maximal value of 0.5 mg. The close correlation between the concentration of nuclear-bound estradiol receptors and the number of initiation sites for RNA synthesis in vivo is at present only a temporal correlation but does raise the possibility that gene transcription in chick oviduct may depend upon the amount of estradiol receptor bound to the target cell nuclei.


Surface morphology and agglutinability with concanavalin A in normal and transformed murine fibroblasts.

The surface morphology of attached and suspended normal and transformed fibroblasts has been studied with the scanning electron microscope. Normal murine fibroblasts (3T3) grow in vitro with widely extended leading lamellae. During most parts of the cell cycle the surfaces of these cells are practically free of microvilli. When the cells round up for mitosis, their cell surfaces become adorned with many microvilli. In contrast, simian virus 40-transformed fibroblasts (SV3T3) grow more compact, and their cell surfaces remain smooth throughout the life cycle. When confluent 3T3 and SV3T3 cells are suspended with ethylenediaminetetraacetic acid (EDTA) for agglutination assays, similar differences in surface morphology are found: 3T3 cells always bear many microvilli, whereas most SV3T3 cells are essentially free of microvilli. The addition of concanavalin A (Con A) does not influence the surface morphology of the suspended cells. The morphological differences described here may be important for the agglutination process of the normal and transformed 3T3 cells, because they affect the real cell surface area and thus the density of Con A-binding sites.


Characterization of nuclear membranes and endoplasmic reticulum isolated from plant tissue.

Nuclei, nuclear membranes and rough endoplasmic reticulum (rER) were isolated from onion root tips and stems. Structural preservation and purity of the fractions was determined by electron microscopic and biochemical methods. Gross compositional data (protein, phospholipid, nonpolar lipids, sterols, RNA, DNA), phospholipid and fatty acid patterns, enzyme activities (ATPases, ADPase, IDPase, glucose-6-phosphatase, 5'-nucleotidase, acid phosphatase, and NADH- and NADPH-cytochrome C reductases), and cytochrome contents were determined. A stable, high salt-resistant attachment of some DNA with the nuclear membrane was observed as well as the association of some RNA with high salt-treated nuclear and rER membranes. The phospholipid pattern was identical for both nuclear and rER membranes and showed a predominance of lecithin (about 60%) and phosphatidyl ethanolamine (20-24%). Special care was necessary to minimize lipid degradation by phospholipases during isolations. Nonpolar lipids, mostly sterols and triglycerides, accounted for 35-45% of the membrane lipids. Sterol contents were relatively high in both membrane fractions (molar ratios of sterols to phospholipids ranged from 0.12 to 0.43). Sitosterol accounted for about 80% of the total sterols. Palmitic, oleic, and linoleic acids were the most prevalent acids in membrane-bound lipids as well as in storage lipids and occurred in similar proportions in phospholipids, triglycerides and free fatty acids of the membrane. About 80% of the fatty acids in membrane phospholipids and triglycerides were unsaturated. A cytochrome of the b5 type was characterized in these membranes, but P-450-like cytochromes could not be detected. Both NADH and NADPH-cytochrome c reductases were found in nuclear and rER membranes and appeared to be enriched in rER membranes. Among the phosphatases, Mg2+-ATPase and, to lesser extents, ADPase, IDPase and acid phosphatase activities occurred in the fractions, but significant amounts of monovalent ion-stimulated ATPase, 5'-nucleotidase and glucose-6-phosphatase activities did not. The results obtained emphasize that the close biochemical similarities noted between rER and nuclear membranes of animal cells extend to these fractions from plant cells.


Lanthanum: inhibition of ACTH-stimulated cyclic AMP and corticosterone synthesis in isolated rat adrenocortical cells.

Lanthanum (La+++) is a well-known Ca++ antagonist in a number of biological systems. It was used in the present study to examine the role of Ca++ in the regulation of adenyl cyclase of the adrenal cortex by ACTH. In micromolar concentrations, .La+++ inhibited both cyclic AMP and corticosterone response of isolated adrenal cortex cells to ACTH. However, a number of intracellular processes were not affected by La+++. These include the stimulation of steroidogenesis by dibutyryl cyclic AMP, conversion of several steroid precursors into corticosterone, and stimulation of the latter by glucose. Thus, inhibition of steroidogenesis by La+++ appears to be solely due to an inhibition of ACTH-stimulated cyclic AMP formation. Electron microscope examination showed that La+++ was localized on plasma membrane of the cells and did not appear to penetrate beyond this region. Since La+++ is believed to replace Ca++ at superficial binding sites on the cell membrane, it is proposed that Ca++ at these sites plays an important role in the regulation of adenyl cyclase by ACTH. Similarities in the role of Ca++ in "excitation-contraction" coupling and in the ACTH-adenyl cyclase system raise the possibility that a contractile protein may be involved in the regulation of adenyl cyclase by those hormones which are known to require Ca++ in the process.


Morphological changes in the neuritic growth cone and target neuron during synaptic junction development in culture.

Our object was to characterize the morphological changes occurring in pre- and postsynaptic elements during their initial contact and subsequent maturation into typical synaptic profiles. Neurons from superior cervical ganglia (SCG) of perinatal rats were freed of their supporting cells and established as isolated cells in culture. To these were added explants of embryonic rat thoracic spinal cord to allow interaction between outgrowing cord neurites and the isolated autonomic neurons. Time of initial contact was assessed by light microscopy; at timed intervals thereafter, cultures were fixed for electron microscopy. Upon contact, growth cone filopodia became extensively applied to the SCG neuronal plasmalemma and manifested numerous punctate regions in which the apposing plasma membranes were separated by only 7-10 nm. The Golgi apparatus of the target neuron hypertrophied, and its production of coated vesicles increased. Similar vesicles were seen in continuity with the SCG plasmalemma near the close contact site; their apparent contribution of a region of postsynaptic membrane with undercoating was considered to be the first definitive sign of synapse formation. Tracer work with peroxidase and ferritin confirmed that the traffic of coated vesicles within the neuronal soma is largely from Golgi region to somal surface. Subsequent to the appearance of postsynaptic density, the form and content of the growth cone was altered by the loss of filopodia and the appearance of synaptic vesicles which gradually became clustered opposite the postsynaptic density. As the synapse matured, synaptic vesicles increased in number, cleft width and content increased, presynaptic density appeared, branched membranous reticulum became greatly diminished, and most lysosomal structures disappeared. Coated vesicles continued to be associated with the postsynaptic membrane at all stages of maturation. The incorporation of Golgi-derived vesicles into discrete regions of the cell membrane could provide the mechanism for confining specific characteristics of the neuronal membrane to the synaptic region.


Rapid breakdown of diphosphoinositide and triphosphoinositide in erythrocyte membranes.

Incubation of rabbit erythrocytes with 32Pi resulted in labeling of membrane diphosphoinositide, triphosphoinositide, and phosphatidic acid. Hypotonic lysis at 37 degress C resulted in an extremely rapid breakdown of the labeled polyphosphoinositides. This breakdown could be retarded by lysis in the presence of EDTA and by lowering the temperature to 0 degrees thus allowing preparation of membranes with minimum breakdown of the labeled lipids. Rapid breakdown of di- and triphosphoinositide in isolated membranes could be initiated by Ca++ or to a lesser extent by Mg++ and prevented by detergents and by heating to 75 degrees C. Assay of radiolabeled lipid was carried out by a method which bypassed prior lipid extraction and which enabled sequential sampling of reactions at 10-second intervals. This method was more convenient than standard procedures and gave yields of di- and triphosphoinositide equivalent to that obtained by the method of Folch.


Light-microscopic observations of individual microtubules reconstituted from brain tubulin.

The course of polymerization of individual brain microtubules could be observed with a light microscope employing dark-field illumination. Statistical analysis of the increase in microtubule length during the polymerization was in accordance with the time course of viscosity change of the tubulin solution. After a plateau level in viscosity was attained, there was no significant change in histograms showing length distribution. These observations were confirmed with fixed and stained microtubules, using a phase-contrast microscope. Observations with dark-field illumination revealed that reconstituted microtubules depolymerized and disappeared immediately upon exposure to buffer containing CaCl2 or sulphydryl reagents such as p-chloromercuriphenyl sulphonic acid (PCMPS) and p-chloromercuribenzoic acid (PCMB). They were also cold-labile. The growth of heterogeneous microtubules which were assembled by mixing purified tubulin dimers with ciliary outer fibres could also be followed with these optical systems.


Trypsinized BHK21 cells aggregate in the presence of metabolic inhibitors and in the absence of divalent cations.

The rapid formation of adhesions in suspension by lightly trypsinized BHK21 cells is not dependent on protein synthesis, and only in part on cellular metabolism, although it is completely inhibited by heat- and aldehyde-fixation of the cells. A requirement for protein synthesis becomes evident only if cells are exposed to high levels of trypsin for long periods. Formation of adhesions does not require addition to the medium of divalent cations, although it is increased by divalent manganese and cobalt ions. It is promoted by cytochalasin B and by cyclic AMP and is not inhibited by p-mercuriphenylsulphonate. We discuss a possible relationship between aggregation and the formation of gap junctions.


[Determination of vitamin D3 and its isomers in D3 resins and in mixtures with vitamin A acetate (author's transl)].

Vitamin D3 and its isomers, prepared from 7-dehydrocholesterol by photochemical reaction, can be separated and quantitatively determined by high-pressure liquid chromatography on aluminium oxide using chloroform as the eluting agent. The determination of small amounts of vitamin D3 in an excess of vitamin A acetate can be achieved by the same method.


Specific thin-layer chromatographic method for the determination of theophylline in plasma in the presence of some other drugs.

A simple spectrodensitometric method for the direct determination of theophylline was developed from measurement of the absorbance of the compound on silica gel layers irradiated at 275 nm. Auantities as low as 0.010 mug can be detected and a linear relationship was obtained between peak area and the amount of the drug in the spots from 0.025-0.200 mug. The recovery over the usual range of plasma concentration (2.5-20 mug/ml) was 95-107%. The method is sufficiently sensitive and specific for clinical purposes and the time for the assay is about 2 h. Caffeine, frequently present in human plasma, was well separated from theophylline at all concentration levels as were several other drugs commonly used in respiratory problems.


The separation by thin-layer chromatography of trace metals as their tetraphenylporphyrin chelates.

The tetraphenylporphyrin chelates of iron, cobalt, manganese, nickel, zinc, copper, rhodium lead, cadmium and mercury were prepared and their chromatographic behaviour investigated using four different adsorbents and eleven different solvent systems. Mixtures of lead, cadmium and mercury chelates exhibited isographic behaviour in all conditions; mixtures of the other metal chelates were adequately resolved on silica (MN Polygram Sil SHR) by the solvent system light petroleum (b.p. 80-100 degrees)-toluene-acetic acid-water (66:33:85:15, v/v, upper phase). The intense colours of the chelates permit their visual detection at levels of about 10(-10) moles; using the integrated ion-current mass spectrometric procedure, as little as 10(-14) moles of metal could be detected and assessed.


Separation of C27, C28 and C29 sterols by reversed-phase high-performance liquid chromatography on small particles.

The application of reversed-phase high-performance liquid chromatography to the analytical- and preparative-scale separation of sterols has been evaluated. The capacity factors, k', for a number of compounds chromatographed on a muBondapak C18 (LESS THAN 10 MUM) COLUMN ARE PRESENTED. C27, C28 and C29 sterols and also sterols differing in degree of unsaturation could be readily separated as their acetates in this system. The present reversed-phase chromatographic method is apparently not as selective as silver nitrate-silica gel thin-layer chromatography for the position of unsaturation in the sterol molecule.


High-speed liquid chromatography of polychlorinated biphenyls and related compounds.

High-speed liquid chromatography in the system silica gel-dry n-hexane has been used to characterize the behaviour of a series of commercially available mixtures of chlorinated biphenyls, viz., Aroclor 1221-1268. An attempt has been made to assign a large number of peaks in the chromatograms of the Aroclors to the individual constituents. To this end, retention times and UV spectra were recorded for 47 polychlorinated biphenyls. The dependence of retention and spectral characteristics on the chlorine content of the substituted biphenyls is discussed


Plasma levels of beta-MSH and ACTH during acute stresses and metyrapone administration in man.

Simultaneous measurements of both beta-melanocyte stimulating hormone (beta-MSH) and adrenocorticotropic hormone (ACTH) in extracted plasma were performed by specific radioimmunoassays. During insulin-induced hypoglycemia, there was a marked increase of plasma ACTH levels and a slight but significant increase of plasma beta-MSH levels. Lysine-vasopressin on the other hand, caused a significant rise of plasma ACTH levels without corresponding response of plasma beta-MSH. Following glucagon administration, neither hormone rose significantly. However, metyrapone infusion caused a significant increase of both ACTH and beta-MSH levels, and frequent blood sampling revealed that both hormones were secreted episodically, and that peaks generally coincided with each other. These data suggest that the secretion of these two hormones can occur together in most instances, and that the same mechanism is involved in the secretion of both hormones under the negative feedback control.


Calcium and folic acid absorption in patients taking anticonvulsant drugs.

Calcium and folic acid absorption were studied in 28 adult male epileptics on chronic anticonvulsant therapy. In 16 patients on diphenylhydantoin alone, calcium absorption was abnormal in 9. In 12 patients on both diphenylhydantoin and phenobarbital, calcium absorption was abnormal in 3 patients. Folic acid (3H-PGA) absorption was normal in all but one patient, while serum folate (less than 6.4 ng/ml) was reduced in all patients. Hypocalcemia (less than 8.5 mg/100 ml) occurred in only 2 patients, while serum alkaline phosphatase was elevated in 7 patients. These findings support the proposal that rickets and osteomalacia reported in patients on chronic anticonvulsant therapy results from reduced calcium absorption. The effect of these drugs appears to be the acceleration of the metabolism of vitamin D and an increase in the excretion of polar metabolites. This may result in reduced levels of 25-hydroxycholecalciferol and 1,25-dihydroxycholecalciferol which are necessary for normal absorption of calcium. Since calcium absorption may be impaired secondary to a relative vitamin D deficiency, a supplemental increase in vitamin D intake by patients on anticonvulsant drugs is recommended.


Sensitivity of infectious bovine rhinotracheitis virus to ether.

The sensitivity of 12 field isolates of infectious boviine rhinotracheitis (IBR) virus and four commercial modified-live infectious bovine rhinotracheitis virus vaccine strains was determined after exposure to 20% ethyl ether (anesthetic) for 16 h at 4 c. The infectivity of five field strains was reduced by varying degrees, whereas seven were found to be resistant. Three vaccine strains were moderately sensitive, and one strain was resistant. Four of the sensitive field strains were isolated from the conjunctiva and the other was isolated from the surface of the epiglottis of natural infective cattle. Strains of virus isolated from fetal tissue and nasal cavity were resistant. All viruses were readily inactivated by chloroform treatment.


Epidemiologic study of a leukocyte-transforming agent in a general population.

Studies by others have demonstrated a leukocyte-transforming agent(s) (LTA) in the oropharyngeal secretions of a significant number of individuals with Epstein-Barr virus-associated infectious mononucleosis, cancer, and, to a lesser extent, an outpatient population. This present study determines by systematic sampling the prevalence of LTA in 27 families of a semirural community. Throat swab inoculums from three of 54 adults and none of 44 children induced transformation of umbilical cord lymphocytes. Complement-dependent Epstein-Barr virus nuclear antigen was detected in two of the three transformed cell cultures. The three LTA-positive individuals were characterized by the absence of serologic evidence of a recent Epstein-Barr virus infection and the lack of elevated antibodies against the viral capsid antigen of Epstein-Barr virus.


Intrinsic mineralocorticoid agonist activity of some nonsteroidal anti-inflammatory drugs. A postulated mechanism for sodium retention.

Because some nonsteroidal anti-inflammatory drugs (NSAID) induce salt and water retention and exhibit other steroid-like actions, studies were performed to ascertain whether these drugs possess intrinsic mineralocorticoid agonist activity. In vitro competitive binding assays utilizing tissue from adrenalectomized rats demonstrated that some NSAID can displace [3H]-aldosterone from renal cytoplasmic mineralocorticoid receptors. Displacement potency for these sites was in the sequence: aldosterone greater than spironolactone greater than phenylbutazone (PBZ) greater than aspirin (ASA) greater than indomethacin (IDM). Concentration ratios required to obtain significant displacement of [3H]aldosterone were high but clearly within the therapeutic range for PBZ and ASA but not IDM. The analogues oxyphenbutazone (OBZ) and sodium salicylate (SS) were similar in binding activity to PBZ and ASA, respectively. Lineweaver-Burk analysis revealed that the inhibition of [3H]aldosterone binding was competitive in nature. In addition, PBZ was shown to prevent the nuclear binding of [3H]aldosterone. In vivo injection of PBZ and ASA resulted in competition for [3H]aldosterone renal binding comparable to the in vitro studies. Administration of PBZ and OBZ to adrenalectomized rats resulted in significant salt retention whereas ASA and SS did not differ significantly from controls. Salt retention elicited by PBZ and OBZ was inhibited by spironolactone, a competitive mineralocorticoid antagonist. These data suggest that, despite nonsteroidal structures, PBZ and OBZ induce salt retention via a receptor-mediated mineralocorticoid pathway analogous to aldosterone action.


Hormone-sensitive fat cell adenylate cyclase in the rat. Influences of growth, cell size, and aging.

The possibility has been explored that decreases of adenylate cyclase may explain diminished hormone sensitivity of adipose tissue with aging. Isolated cells were prepared from epididymal fat pads of rats 1-, 2-, 6-, 12-, and 24-mo old, fixed in OSO4, and sized and counted with a Coulter apparatus. Adenylate cyclase was assayed in cell membranes (ghosts) using [alpha-32P] ATP as substrate and expressed as cyclic [32P] AMP/10 min per mg protein or per 10(6) cells. Enzyme activity was determined for the basal state and in the presence of varying concentrations of glucagon, ACTH, epinephrine, and fluoride. Basal activity per cell increased in threefold between 1 and 2 mo with a comparable increase in cell surface area, suggesting synthesis of enzyme along with new cell membrane. Although epinephrine stimulated adenylate cyclase 8-fold and fluoride 12-fold throughout the life-span of the rat, stimulated activity paralleled basal levels, decreasing 60% between 2 and 24 mo per mg protein and 40% between 6 and 24 mo per cell. Glucagon stimulated adenylate cyclase 4.5-fold relative to basal in the 1-mo-old rat, but its effect then rapidly decreased and was absent by 12 mo. The fourfold stimulation by ACTH noted in the 1-mo-old animals decreased gradually with age but was still twice basal at 24 mo. Since no significant change of cell size occurred after 6 mo, diminished hormone sensitivity with senescence cannot be related to cell size. Similar age-related patterns of hormonal activation were evoked by 5'-guanylyl-imidodiphosphate [GMP-P(NH)P], a nucleotide analogue which increased both basal- and hormone-activated enzyme at all ages studied. Dose-response curves to hormones, fluoride, and GMP-P (NH)P were not affected by age. High Mg++ (50 mM) in the presence of GMP-P-(NH)P stimulated adenylate cyclase to levels greater than with fluoride, but a similar loss of activity with aging was still observed. Loss of hormone receptors may partially explain the age-related decreases of glucagon and ACTH-sensitive adenylate cyclase, but decreased basal-, epinephrine-, fluoride-, and GMP-P-(NH) P-stimulated responses suggest loss of the catalytic component of the adenylate cyclase enzyme complex in the aging fat cell membranes.


Potentiation of erythropoiesis in vitro by dexamethasone.

The effect of dexamethasone on erythropoiesis was examined in vitro. Hematopoietic cells from 13-day mouse fetal livers were cultured for 48 h in the presence or absence of erythropoietin and erythroid colonies enumerated. Colony formation occurring in cultures containing no added erythropoietin was inhibited by the incorporation of antierythropoietin antibody, suggesting that these colonies formed in response to endogenous hepatic erythropoietin. Maximal colony formation was observed with 0.5 U/ml of sheep erythropoietin. Dexamethasone increased erythroid colony formation with peak stimulation at 10(-9) M. Dexamethasone potentiation was most marked in cultures containing less than maximally stimulating concentrations of erythropoietin. The cells required only a brief exposure to glucocorticosteroid to exhibit the augmented cloning capacity, and dexamethasone stimulation was inhibited by progesterone (10(-6) M). A comparable response to dexamethasone was observed in cultures of adult murine and human bone marrow erythroid progenitors, implying that the phenomenon is not peculiar to fetal cells and is not dependent on the presence of fetal hepatocytes. These data suggest that erythroid progenitor cells possess a glucocorticoid receptor mechanism that can modulate the response to erythropoietin in vitro.


Biosynthesis of proinsulin and insulin in newborn rat pancreas. Interaction of glucose, cyclic AMP, somatostatin, and sulfonylureas on the (3H) leucine incorporation into immunoreactive insulin.

The purpose of the present study was to investigate the regulation of insulin biosynthesis during the perinatal period. The incorporation of [3H]leucine into total immunoreactive insulin (IRI) and into IRI fractions was measured by a specific immunoprecipitation procedure after incubation, extraction, and gel filtration in isolated 3-day-old rat pancreases without prior isolation of islets. IRI fractions were identified by their elution profile, their immunological properties, and their ability to compete with the binding of 125 I-insulin in rat liver plasma membranes. No specific incorporation of [3H]leucine was found in the IRI eluted in the void volume, making it unlikely that this fraction behaves as a precursor of (pro) insulin in this system. In all conditions tested, the incorporation of [3H]leucine was linearly correlated with time. Optimal concentration of glucose (11 mM) activated six- to sevenfold the [3H]leucine incorporation into IRI. Theophylline or N6O2-dibutyryl- (db) cAMP at 1.6 mM glucose significantly increased the [3H]leucine incorporation. Glucose at 16.7 mM further enhanced the effect of both drugs. Contrarily, somatostatin (1-10 mug/ml) inhibits the rate of [3H]leucine incorporation into IRI in the presence of 11 mM glucose; this effect was observed at 5.5 mM glucose and was not modified by any further increase in glucose concentrations up to 27.5 mM. Theophylline or dbcAMP at 10 mM concentration did not reverse the somatostatin inhibitory effect on either insulin biosynthesis or release. Somatostatin also inhibited both processes in isolated islets from the 3-day-old rat pancreas. High Ca++ concentration in the incubation medium reversed the inhibitory effect of somatostatin on glucose-induced insulin biosynthesis as well as release. In both systems the inhibitory effect of somatostatin on insulin biosynthesis and release correlated well. Glipizide (10-100 muM) AND TOLBUTAMIDE (400 MUM) inhibited the stimulatory effect of glucose, dbcAMP, and theophylline on [3H]leucine incorporation into IRI. The concentrations of glipizide that were effective in inhibiting [3H]leucine incorporation into IRI were smaller than those required to inhibit the phosphodiesterase activity in isolated islets of 3-day-old rat pancreas. These data suggest the following conclusions: (a) the role of the cAMP-phosphodiesterase system on insulin biosynthesis is likely to be greater in newborns than in adults; (b) the greater effectiveness of glucose and the cAMP system on insulin biosynthesis than on insulin release might possibly be related to the rapid accumulation of pancreatic IRI which is observed in the perinatal period; (c) somatostatin, by direct interaction with the endocrine tissue, can inhibit glucose and cAMP-induced insulin biosynthesis as well as release; calcium reverses this inhibition; (d) sulfonylureas inhibit insulin biosynthesis in newborn rat pancreas an effect which has to be considered in the use of these agents in human disease.


Metabolism and biological activity of parathyroid hormone in renal cortical membranes.

Recent studies from several laboratories have documented the presence of fragments of parathyroid hormone in blood or peripheral tissues or in both. Inasmuch as amino-terminal fragments are known to be biologically active, it has been suggested that fragments, rather than the intact polypeptide of 84 amino acids, might be the active molecular species in tissue fluids. Accordingly, the metabolism of native bovine parathyroid hormone, bPTH-(1-84), was studied in purified renal cortical membranes from several species and correlated with hormonal stimulation of adenylyl cyclase in these membranes in vitro. Analysis of whole incubation mixtures or membrane-bound hormone by gel electrophoresis and gel chromatography after incubation of [3H]bPTH-(1-84) or 125-I-labeled bPTH-(1-84) or unlabeled biologically active bPTH-(1-84) with purified canine renal cortical membranes revealed no evidence of proteolysis, and yet the uncleaved hormone readily stimulated adenylyl cyclase. Kinetic studies of hormone-stimulated adenylyl cyclase activity revealed no difference in rate of onset of activity between bPTH-(1-84) And the active synthetic amino-terminal tetratriacontapeptide bPTH-(1-34), and hence there was no evidence of precursor-product relationship between the native hormone and an active amino-terminal fragment. The results suggest, insofar as the activity detected in these membranes reflects the biological response of the hormone in vivo, that the native hormone is indeed biologically active at the receptor level directly without the requirement for cleavage into active fragments.


Abnormal properties of collagen lysyl hydroxylase from skin fibroblasts of siblings with hydroxylysine-deficient collagen.

Skin fibroblasts from two siblings with hydroxylysine-deficient collagen collagen (Ehlers-Danlos syndrome, type VI) contained normal levels of collagen prolyl hydroxylase activity but were markedly deficient in collagen lysyl hydroxylase activity. The deficiency was evident in all fractions of cell lysates, in low and high ionic strength buffers, and in detergent. Assays of mixtures of wild-type and mutant cell lysates indicated no activation of mutant enzyme by factors in wild-type cells or inhibition of normal enzyme by material in mutant cells. Wild type or mutant cells cultured with ascorbic acid (50 mug/ml of culture medium, added daily) contained approximately the same level of lysyl hydroxylase activity as cells cultured without ascorbate, but prolyl hydroxylase activity without ascorbate was depressed in both an average of 41%. The mutant lysyl hydroxylase was less stable at 37 degrees C than the wild type and did not form high molecular weight aggregates in low ionic strength buffers, as did the control enzyme. The activity of the mutant enzyme was maximally stimulated after dialysis against buffer solutions containing 10 mM dithiothreitol. When  assayed in 100 muM dithiothreitol, the mutant enzyme exhibited a higher apparent Km for ascorbate (20 muM) than the wild type (4 muM). In 1.0 mM dithiothreitol the mutant enzyme's apparent Km for ascorbate was reduced to 5 muM. Wild type and mutant enzymes had the same apparent Km for alpha-keto-glutarate (20 muM). The properties of prolyl hydroxylase in wild type and mutant cells were identical: apparent Km's for ascorbate and alpha-ketoglutarate were 100 muM and 20 muM, respectively. If mutant enzyme protein with altered kinetic properties is the only enzyme functioning to hydroxylate lysyl residues in collagen, the variations in hydroxylysine content observed in collagen from different tissues in the subjects reported here could be in part due to differences in cofactor concentrations and in rate and sequence of events in collagen synthesis in different tissues.


Development and evaluation of porous dental implants in miniature swine.

Organized bone ingrowth in endosteal porous implants fabricated from VMC titanium alloy and surgically implanted with a tight interference fit, securely anchored the implants in fresh and healed mandibular premolar sites of miniature swine. This bone-implant union retained its integrity under high as well as slight masticatory stresses up to one-year after implantation. Bone invasion of the alumina porcelain implants was impeded by the lack of adequate interconnecting porosity; when the porosity was increased, insufficient ceramic strength prohibited a tight initial bone-implant fit. As a consequence, inadequate initial implant stability resulted in a soft tissue encapsulation of the majority of the ceramic implants. Histological examination and mechanical testing results were similar for bone-ingrown implants exposed to different experimental stresses for 4, 5, 8, and 12 months. Bone ingrowth and interface shear strengths were also similar in the different VMC pore sizes and shapes investigated. The design of intraoral attachments appeared critical, at least in swine where no postoperative treatment was administered. Gingival inflammation and alveolar bone resorption caused by calculus were severe around truncated cone-shaped devices. Slender transgingival posts, occlusal caps, and crown restorations were less susceptible to calculus accumulation, resulting in a more satisfactory gingival and subgingival response. Excessive epithelial invagination was a problem only in implants with transgingival truncated cones. Good adherence of soft tissue to metal under the gingival mucosa prevented epithelial migration around implants with other transgingival devices. Alveolar bone resportion around the tops of bone-ingrown implants was minimal at the time intervals examined (up to one year); however, a definite conclusion should be delayed until longer-term implants under full occlusion are evaluated.


Origin, diagnosis, and treatment of the dental manifestations of vitamin D-resistant rickets: review of the literature and report of case.

Previous discussions center on early diagnosis, initial treatment, and follow-up therapy for the patient with vitamin D-resistant rickets. Both the medical and dental aspects of treatment for these patients has a long-range effect on the normal developmental patterns. Although treatment is begun at an early age, some rachitic skeletal effects such as minor bowing of the legs and bossing of the skull will invariably be noticed. In patients with controlled rickets the alveolar processes undergo normal development, with apparent normal dental eruption. The poor development and calcification of the alveolus seen in the untreated patient leads to loss of the lamina dura and periodontal ligament of the teeth. Patients with resistant rickets possess a functional dentition, although not without inherent defects. Various degrees of fracture and attrition of enamel can be seen, and hypoplasia of dentin is nearly a universal result. Defects extending to the dentinoenamel junction have been shown in repeated cases. Cementum, because of its close relationship with dentin calcification, also appears abnormal. Pulp tissue may undergo abberations of physiology in resistant rickets, although further work in this respect is needed. With respect to the possible dental pathoses seen in this disease, the dental history of the patient with resistant rickets discussed in this report showed that several of the deciduous teeth, possibly the mandibular left second premolar and right first molar, and definitely the maxillary right second premolar and canine and the mandibular left canine had all undergone pulpal degeneration of apparently unknown causation. In the maxillary right second premolar and the mandibular left canine, enamel fractures were clinically and radiographically apparent. However, the maxillary right canine originally had an acute abscess with no defects other than normal, minimal wear facets. No causative factor for its necrosis could be found. Overt enamel fractures in the maxillary right second premolar and the mandibular left canine may have led to microexposures of the pulp with subsequent bacterial pulpal contamination. Suppuration present in several of the pulps when first entered during endodontic treatment, as well as chronic fistulas in several areas, support the conclusion that contamination by some means does indeed occur.


An action of disodium cromoglycate: inhibition of cyclic 3',5'-AMP phosphodiesterase.

Disodium cromoglycate (DSCG) prevents allergic asthma by inhibiting the release of chemical mediators of immediate-type allergic reactions. The mechanism of this action is unclear and prompted us to examine the effect of DSCG on cyclic adenosine 3',5'-monophosphate (cAMP), the implicated regulator of IgE-mediated reactions. We used the peripheral blood lymphocyte as a model to mirror the biochemical events occurring in the allergic shock organs. Isolated peripheral blood lymphocytes from perennial allergic asthmatic children receiving only DSCG had significantly (p less than 0.005) lower phosphodiesterase (PDE) activity (mean 1.05 +/- 0.17 SE per 10(6) cells) than normal individuals (2.93 +/- 0.14) and allergic children receiving methylxanthines (4.08 +/- 0.28) or no medications (3.58 +/- 0.2). DSCG (10 mug/ml) significantly lowered PDE activity in normal lymphocytes (p less than 0.005) in a beef heart extract (p less than 0.001), and 100 mug/ml lowered PDE activity in fetal rabbit lung homogenates (p less than 0.001). DSCG (10 mug/ml) significantly elevated (p less than 0.01) cAMP concentration in normal human lymphocytes (118 +/- 38 vs 30 +/- 10 picomoles cAMP/10(6) lymphocytes). Thus, DSCG appears to inhibit chemical mediator release by increasing intracellular cAMP through the inhibition of cAMP PDE.


The behaviour of porcine cytomegalovirus in commercial pig herds.

A longitudinal, virological and serological study of pigs in two herds with respiratory disease showed that infection by porcine cytomegalovirus (PCMV) was universal in both. Virus excretion usually began when piglets were between 3 and 6 weeks of age and reached a maximum between 5 and 8 weeks; it was usually no longer detectable at 11-12 weeks. Antibody demonstrable in indirect immunofluorescence (IIF) tests was present to moderate or high titre in all piglets at 2-3 weeks. This was presumed to be maternal in origin as it declined in titre between 2-3 and 5-6 weeks. After this fall the majority of piglets showed seroconversion as a result of virus infection. One group of 12 pigs in which infection occurred earlier than usual  showed a very poor antibody response, which, nevertheless, persisted through to week 27. The findings are discussed with relation to porcine atrophic rhinitis and cytomegalovirus infection in other species.


Rhinoviruses in Britain 1963-1973.

The serological examination of over 900 rhinovirus strains isolated in Britain over a 10-year period has demonstrated a wide variety of different serotypes. Their seasonal and temporal distribution are described and discussed.


Enhancement of reaginic and hemagglutinating antibody production by an extract of Bordetella pertussis containing histamine sensitizing factor.

The effect of an extract containing the histamine-sensitizing factor (HSF) of Bordetella pertussis on the immune response of mice to ovalbumin was investigated with respect to dose of antigen and adjuvant. Of particular interest was the enhancement of reaginic antibody production. In comparison to the Al(OH)3 induced production of reaginic antibody where low doses of antigen and adjuvant yield high titers of reagin, the HSF extract demonstrated optimal adjuvant activity at high doses of both adjuvant and antigen. The reaginic antibody response was maximal usually by 2 to 3 weeks post-immunization and persisted for long periods of time. The hemagglutinating antibody response was maximal at 8 to 10 weeks post-immunization. The initial treatment of mice with HSF extract plus antigen resulted in the production of memory cells since a subsequent immunization with ovalbumin alone evoked a secondary reaginic response. These observations may have implications in clinical allergy since substances similar to the pertussis factor might be produced by other microbial organisms and these substances could modulate the immunologic response of individuals to common allergens.


Antibody-dependent cell-mediated cytotoxicity to target cells infected with type 1 and type 2 herpes simplex virus.

The phenomenon of antibody-dependent cell-mediated cytoxicity (ADCC) has been extended to include target cells acutely infected with herpes simplex type 1 virus (HSV-1) or herpes simplex type 2 virus (HSV-2) in an in vitro system that employs immune human serum and human blood mononuclear cells. The cytotoxic reaction was detectable after 1 hr of incubation and was complete between 4 and 8 hr. The amount of ADCC noted was directly proportional to the logarithm(10) of the effector: target cell ratio (E:T), and ADCC was noted at E:T as low as 1:1. The mononuclear effector cell was present in the blood of both HSV immune and non-immune individuals. The immune serum factor was demonstrated to be an antibody with specificity for HSV membrane antigen(s) and was reactive with target cells infected with either of the two HSV types. The antibody rendered the mononuclear cell cytotoxic by sensitization of the target cell rather than by direct attachment to or "arming" of the mononuclear cell. The physiochemical properties of the antibody as well as its presence in cord blood demonstrated that it is an immunoglobulin on the IgG class.


Mechanism of immunologic resistance to herpes simplex virus 1 (HSV-1) infection.

Susceptibility of adult mice to i.p. infection with HSV-1 was greatly increased by administration of a single dose of cyclophosphamide. Mortality of cyclophosphamide-treated virus-infected mice was associated with increased virus replication and pathologic changes in brain and liver. The development of a fatal infection in immunosuppressed mice could be curtailed after transfer of specifically immune spleen cells. Passively transferred antibody had no such effect. Protective activity of spleen cells was significantly reduced after pretreatment with anti-theta serum. Significant protection was also achieved when normal spleen cells plus immune serum were administered simultaneously. Our results indicate that protection against this virus infection is predominantly T cell dependent, and suggests that antibody-dependent cell-mediated protection may also be operative in vivo.


Adenosine 3'-5'-monophosphate-dependent protein kinases of neonatal human skin.

Three forms of adenosine 3'-5'-cyclic monophosphate-dependent protein kinases have been isolated from neonatal human skin. An analysis of their properties indicates that they resemble those of cyclic AMP-dependent protein phosphotransferases of adult skin.


Diarrhea caused in gnotobiotic piglets by the reovirus-like agent of human infantile gastroenteritis.

One- to four-day-old gnotobiotic piglets were inoculated orally with a reovirus-like agent obtained from human infants with acute gastroenteritis. Diarrhea developed in the piglets two to seven days after inoculation and was reproduced for five serial passages in one sequence and for three passages in another. Nineteen of 21 inoculated piglets developed diarrhea; reovirus-like particles were observed in intestinal contents and/or fecal samples from 17 animals with illness and from two inoculated piglets that did not develop diarrhea. One piglet, for which daily fecal samples were examined by electron microscopy, shed the largest number of virus particles at the onset of diarrhea. Immunofluorescent antibody responses to the reovirus-like agent were detected in sera from the seven inoculated animals that were tested.


Transmission of experimental rhinovirus colds in volunteer married couples.

Communicability of rhinovirus type 16 or type 55 was studied in 24 childless couples; on partner (the donor) was infected with laboratory-grown virus. Initially, both partners lacked antibody to the challenge agent. Rates of transmission between partners were 41% and 33% for type 16 and type 55, respectively. These rates are similar to those determined in epidemiologic studies of natural rhinovirus infection. Although the mucosa of the anterior nares was shown to be highly susceptible to infection (less than one 50% tissue culture infective dose [TCID50]), transmission rarely occurred unless (1) at least 1,000 TCID50 of virus was recovered from the donor's nasal washing, (2) the donor had virus on his hands and anterior nares, (3) he was at least moderately symptomatic, and (4) he spent many hours with his spouse. Since person-to-person transfer of rhinovirus was so dependent upon time spent together and shedding of large amounts of virus by the donor, it seems possible that the chain of infection could be interrupted by environmental manipulation.


A rhesus monkey model for the study of Bolivian hemorrhagic fever.

Two groups of rhesus monkeys were inoculated with either 10(5) (group 1) or 10(3) (group 2) plaque-forming units of Machupo virus, the etiologic virus of Bolivian hemorrhagic fever. The monkeys were observed for clinical signs; body temperatures, viremias, hematologic changes, and virus-neutralizing antibody were measured. The onset of clinical signs for groups 1 and 2 occurred on days 4-6 and 7-10, respectively, with fever, anorexia, and depression. These and other signs became more severe, and all of the monkeys died; the respective mean times to death for groups 1 and 2 were 14.3 and 19.5 days. Hematocrit, neutrophil, and lymphocyte values decreased in both groups until a few days before death and then increased slightly. Viremias in the two groups peaked on days 13 and 16, respectively, and persisted until death; the sole exception was one monkey in group 2 that developed neutralizing antibody by day 21. The response of the rhesus monkey to Machupo virus thus provides a useful model for the study of Bolivian hemorrhagic fever.


Synergistic antiviral effects of adenine arabinoside and humoral antibodies in experimental encephalitis due to Herpesvirus hominis.

The antiviral effects of humoral antibodies and adenine arabinoside on encephalitis due to Herpesvirus hominis were studied in three-week-old mice. Exogenously administered antibodies to H. hominis, of rabbit or human origin, significantly reduced morbidity and mortality rates from H. hominis encephalitis if enough antibodies were given during the early phase of infection. Adenine arabinoside could also modulate the pathogenesis and reduce the mortality rate in mice with H. hominis encephalitis. Simultaneous administration of adenine arabinoside and human immune globulin resulted in an enhanced protection against H. hominis encephalitis. This increased protection was manifested by a significant reduction of mortality rate, a decrease in concentration of virus, and a lessening of histopathologic damage in the brain tissues. Mechanisms involved in the enhanced protective effects were not well defined. The use of adenine arabinoside plus human immune globulin did not completely suppress viral replication. Therefore, host recovery was probably mediated through (1) partial suppression of viral replication by adenine arabinoside, (2) neutralization of cell-free virus by antibodies, and (3) collaboration of antibodies with other host resistance factors (e.g., complement, leukocytes, nonimmune effector cells, etc.). Our data suggest that control of severe H. hominis infection may require the combined use of an antiviral agent and humoral factor and, perhaps, enhancement of host responses by other means.


Clinical efficacy of adenine arabinoside in therapy of cytomegalovirus infections in renal allograft recipients.

Three renal allograft recipients, two with cytomegalovirus pneumonia and one with cytomegalovirus retinitis, were treated with adenine arabinoside. The dose was 5-10 mg/kg per day administered for four to six days. There was no clinical improvement in any of the patients. One patient died from overwhelming interstitial viral pneumonia. Cytomegalovirus was readily isolated from two patients after therapy (autopsied lung of one and the saliva of the other). Suppression of viruria was observed and quantitated in one patient (10(4.0)-10(2.5) 50% tissue culture infective doses/0.2 ml). However, the suppression was transient, and viral titers returned to the levels before therapy within two months after completion of therapy. Treatment was stopped in two cases when hematologic toxicity (drop in hematocrit and platelet and leukocyte counts) was noted in the patients five days after initiation of therapy with adenine arabinoside. The drug appears to be ineffective and possibly toxic in immunosuppressed renal allograft recipients infected with cytomegalovirus.


An improved method for the extraction of endogenous platelet serotonin.

Investigations of blood platelet serotonin content, turnover, metabolism, and secretion require sensitive methods for accurate measurement of the amine. The present study has sought a simple, efficient procedure for extracting total platelet serotonin which could also be used for analysis of other platelet chemical constituents. ZnSO4 precipitation, the standard technique for extracting 5-HT, failed to recover total platelet serotonin despite physical and chemical manipulations to improve its availability. Perchloric acid extraction was found to be more efficient, resulting in significantly increased values for endogenous platelet serotonin and recovery of 97 per cent of standard amounts of 5-HT added to platelet pellets. In addition, the perchloric acid extracts could be used for analysis of other platelet constituents such as adenine nucleotides. The improved procedure eliminates the need for large numbers of replicate samples for several assays and, at the same time, provides a more sensitive method for recovery of total platelet serotonin. It may also prove valuable for prolonged in vitro experiments in which extraction must be followed by additional isolation procedures in order to separate indole metabolites of serotonin from the amine for specific quantitation.


Effects of chronic uremia, hemodialysis, and renal transplantation on plasma lipids and lipoproteins in man.

Since quantitative and qualitative alterations in plasma lipoproteins may provide insights into mechanism(s) of altered lipid transport in renal failure, whole plasma triglyceride (TG) and cholesterol (Chol) concentrations and lipoprotein neutral lipids and composition were examined in patients with chronic renal failure (undialyzed and dialyzed) and following successful renal transplantation. Both uremic groups demonstrated increased TG (p less than 0.001) and normal Chol in whole plasma and increased total TG and Chol in the very low-density lipoprotein fraction (VLDL). All hyperlipidemic subjects showed a Type IV phenotype. The percentage triglyceride in VLDL was slightly higher than control in the dialysis patients, and significantly increased in LDL in both undialyzed (p less than 0.001) and dialyzed (p less than 0.005) uremic groups. Transplant patients had significant increases (p less than 0.001) in both TG and Chol in whole plasma, and increased total TG and Chol in both the low-density lipoproteins (LDL) and VLDL fractions. Transplant patients with hyperlipidemia showed a variety of phenotypes and an enrichment of triglyceride in VLDL and LDL. These findings indicate that abnormalities in lipoprotein metabolism in renal failure patients are not appreciably affected by chronic dialysis treatment and continue following successful transplantation. The tendency toward increased VLDL and LDL triglyceride content in these patients resembles the lipoprotein neutral lipid composition found in nonrenal patients with similarly elevated plasma lipids. These alterations could result from primary disturbances in VLDL production and/or removal.


Recent advances in our understanding of the vitamin D endocrine system.

Vitamin D3 gives rise to at least one hormone in which the kidney is utilized as an endocrine system. This hormone arises from 25-OH-D3 which in turn is synthesized in the liver from vitamin D3. The production of this calcium and phosphorus mobilizing hormone, namely 1,25-(OH)2D3, is strongly regulated by the need for calcium and phosphorus. The regulation of its production can occur only after initial 1,25-(OH)2D3 is made and brings about the appearance of 25-OH-D3-24hydroxylase. The need for calcium brings about a stimulation of parathyroid hormone secretion. The parathyroid hormone suppresses the 24-hydroxylase and stimulates the 1-hydroxylase. Alternatively, the need for phosphorus directly stimulates the 1-hydroxylase and suppresses the 24-hydroxylase. The 24-hydroxylation appears to be the initial reaction leading to the inactivation and excretion of vitamin D whereas the 1-hydroxylation is the reaction bringing about the activation of the molecule to 1,25-(OH)2D3. The 1,25-(OH)2D3, the 25-OH-D3 and an analog of 1,25-(OH)2D3, namely 1alpha-OH-D3, are potentially extremely useful in the treatment of metabolic bone diseases such as renal osteodystrophy, hepatically related disorders of calcium and bone metabolism, hypoparathyroidism, and vitamin D dependency disease. The 1alpha-OH-D3 is effective by virtue of its conversion to 1,25-(OH)2D3. The 25-hydroxylation of both 1alpha-OH-D3 and vitamin D3 itself occurs predominantly in the liver. Finally, it is not entirely settled whether 1,25-(OH)2D3 is active directly in all of the functions of viramin D or whether it must be further converted metabolically. A new metabolic pathway for vitamin D has been discovered in which 1,25-(OH)2D3 loses its 26 and 27 carbons to carbon dioxide, producing an unknown metabolite. It is not certain whether this pathway represents degradation of the 1,25-(OH)2D3 or its further activation.


Activation of renal erythropoietic factor by phosphorylation.

Renal erythropoietic factor (REF) serves as a substrate for a cyclic nucleotide-dependent protein kinase. The phosphorylation of REF is associated with increased erythropoietic activity. Conversely, REF isolated from hypoxic rats is a poor phosphate acceptor. These findings and the presence of a positive correlation between erythropoietin levels and urinary excretion of adenosine 3',5' monophosphate (cyclic-AMP) in anemic individuals suggest that the cyclic-AMP-protein kinase system plays an important role in the activation of REF.


Effect of acute inhibition of adrenocorticotrophin secretion on plasma corticosteroids in the rhesus monkey (Macaca mulatta).

The half-life of plasma cortisol in the rhesus monkey (M. mulatta), determined by two methods, was about 130 min and longer than that in man; it was unaffected by administration of dexamethasone. Dexamethasone (5 mg, i.v.) immediately inhibited the secretion of ACTH from the monkey pituitary. The plasma half-life of NH2-terminal immunoreactive ACTH was found to be about 55 min which was much longer than the biological half-life. The adrenal synthesis of cortisol was inhibited by metyrapone which caused a prompt increase in the plasma concentration of ACTH and 11-deoxycortisol. The hypothalamic-pituitary-adrenal system of the rhesus monkey sedated with phencyclidine hydrochloride responded rapidly to alteration in the level of steroids in the circulation.


Development of a radioimmunoassay for alpha-melanocyte-stimulating hormone in the rat.

A sensitive and specific radioimmunoassay for alpha-melanocyte-stimulating hormone (alpha-MSH) was developed. Extracts of the neurointermediate lobe of the rat produced displacement curves which were parallel to those obtained with synthetic alpha-MSH. The mean immunoreactive alpha-MSH concentration in neurointermediate lobes from normal adult rats was 2768 +/- 200 (S.E.M.) ng/lobe. This accounted for approximately 78% of the MSH activity of the neurointermediate lobe as measured by bioassay. Much lower levels of immunoreactive alpha-MSH were found in the anterior lobe of the rat. Extracts of rat serum and plasma also contained immunoreactive alpha-MSH and the mean level was found to be 237 +/- 20 pg/ml. This was slightly lower than the level measured in rat plasma by bioassay. Increased levels of alpha-MSH were found in plasma of rats 1 and 3 h after a single injection of trifluoperazine and after 1-5 min of ether anaesthesia. These changes were reflected by decreases in the alpha-MSH content of the neurointermediate lobe.


Interrelationships of pituitary and plasma corticotrophin and plasma corticosterone during adrenocortical regeneration in the rat.

Pituitary and plasma ACTH and plasma corticosterone were measured at various times after adrenal enucleation or adrenalectomy. The changes in ACTH could be inversely correlated with those in plasma corticosterone. They could also be prevented by maintenance therapy with the steroid. The results indicate that the corticosteroids control both the release and the synthesis of ACTH but that their effect is predominantly on the release of the hormone.


Cytoplasmic oestrogen receptor complex of female ovine pituitary: changes associated with the reproductive state and oestradiol treatment.

Binding of oestrone and oestradiol to a pituitary cytosol receptor was studied in ovariectomized sheep during the breeding season, during anoestrus and after oestradiol treatment during anoestrus. All sheep were ovariectomized 2 weeks before removal of the anterior pituitary. Oestrogen treatment consisted of the subcutaneous implantation of Silastic capsules containing oestradiol-17beta 3 weeks before ovariectomy. Association constants (KA) for oestrone and oestradiol and the concentration of binding sites for oestradiol varied with changes in reproductive state. Oestradiol bound more tightly and the concentration of available binding sites was lower during the breeding season than during anoestrus. The affinity of oestrone binding was less during the breeding season than during anoestrus, whilst the concentration of binding sites for this steroid did not change with changes in reproductive state. Treatment of anoestrous sheeep with oestradiol increased the KA for oestradiol and decreased the concentration of binding sites. However, no effect of oestradiol treatment was observed with regard to the KA or concentration of binding sites for oestrone. The results suggest that either the pituitary oestrogen cytoplasmic receptor undergoes a conformational change during the breeding season causing oestradiol to be more tightly bound or the onset of the breeding season is accompanied by the appearance of a new receptor which binds to oestradiol with greater affinity. Oestradiol treatment of anoestrous sheep mimics this change.


Motor patterns in the stomatogastric ganglion of the lobster Panulirus argus.

1. Acitivity patterns arising from the thirty cells of the stomatogastric ganglion of Panulirus argus are described for both a semi-intact preparation and an isolated one. 2. The thirty or so cells can be divided so far into two functional groupings: the gastric mill group, with at least ten motor elements, and the pyloric group with at least fourteen. There is some, but not extensive, interaction between groups. 3. The main gastric mill activity is arranged in two sets of elements, each of which is composed of reciprocating elements innervating antagonistic muscles. Thus alternation in activity between the single LC and the two LG neurones results in alternate closing and opening of the lateral teeth; alternation between the four GM and single CP units results in alternate protraction and retraction of the medial tooth. 4. The two sets are phased to each other in such a way that they cause gastric mill teeth to operate effectively to masticate food. 5. The main pyloric activity is arranged in a three-part cycle with each of three sets of units active in sequence. Activity in two PD and one AB unit is followed by bursts in IC and LP units followed in turn by activity in up to seven PY units. Activity in a single VD neurone is locked to this cycle in a more complex pattern.


Kinetics of 3-O-methyl glucose transport in red blood cells of newborn pigs.

The glucose-permeable fetal red cells in the pig are entirely replaced by glucose-impermeable adult red cells within a month after birth. This study investigates the kinetic parameters of the glucose transport mechanism in newborn pig red cells in comparison with immature adult red cells (reticulocytes) as well as the fully matured adult erythrocytes. Influx and efflux of the nonmetabolizable 3-O-methyl glucose (3-O-M-G) in red cells of newborn pigs saturate at high substrate concentrations and exhibit typical Michaelis-Menten kinetics. Km values for efflux are 15.2 and 18.2 mM for 15 and 22 degrees C, respectively. Q10 computed between 10 and 26 degrees is 5.0. The energy of activation for the transport process is 34,000 cal mol-1. The effectiveness of hexoses in competing with 3-O-M-G in efflux is in the following order: D-glucose greater than D-mannose greater than D-fructose greater than D-galactose. Efflux of 3-O-M-G does not increase with 3-O-M-G or D-ribose in the medium and is reduced by 2,4-dinitroflurobenzene (DNFB), p-chloromercuriphenyl sufonic acid (PCMBS), and phloridzin. The reticulocytes are shown to possess a carrier-mediated transport but with a considerably lower transport rate. As the reticulocytes mature into normal red cells, the carrier transport mechanism is lost.


Enzyme activity changes during cyclic AMP-induced stalk cell differentiation in P4, a variant of Dictyostelium discoideum.

The P4 variant of Dictyostelium discoideum is characterized by the production of fruiting structures in which the overall proportion of stalk to spore material is increased, relative to the wild type. The altered morphology of the mutant is due to increased sensitivity to cyclic AMP which promotes stalk cell differentiation. In the presence of 10-4 M-cyclic AMP the entire population of P4 amoebae forms clumps of stalk cells on the surface of the dialysis membrane support. Measurement of changes in activity of a range of developmentally-regulated enzymes during the development of P4 in the presence and absence of cyclic AMP has allowed us to identify three classes of enzyme: (i) Those, such as beta-glucosidase II, trehalose-6-phosphate synthetase and uridine diphosphogalactose-4-epimerase, which are required for the production of spores. (ii) Enzymes, primarily but perhaps not exclusively, required during stalk cell formation. Typical of these are N-acetylglucosaminidase and alkaline phosphatase. (iii) General enzymes, such as threonine dehydrase, alpha-mannosidase and uridine diphosphoglucose pyrophyosphorylase, which are present inboth pre-stalk and pre-spore cells and appear to be necessary for the development of both cell types.


Mutations in Escherichia coli that relieve catabolite repression of tryptophanase synthesis. Mutations distant from the tryptophanase gene.

Two mutants are described in which the synthesis of tryptophanase is unusually insensitive to catabolite repression. Neither mutation is linked by transduction to the tryptophane structural gene, neither mutation renders the synthesis of beta-galactosidase insensitive to catabolite repression, and the mutations do not permit tryptophanase to be synthesized in strains deficient in adenyl cyclase. During growth in glucose-minimal medium the mutants maintained a similar intracellular concentration of cyclic AMP to their wild-type parent; but since in the wild type the concentration of cyclic AMP was the same in glycerol-minimal medium as in glucose-minimal medium, it is doubtful whether catabolite repression is mediated by measurable changes in the concentration of this nucleotide.


Unique virus-related DNA sequences in sheep progressive pneumonia lung.

Hybridization studies with [3H]-cDNA of progressive pneumonia, maedi and visna viruses demonstrate that lung DNA from sheep afflicted with progressive interstitial pneumonia possesses virus-related sequences not present in normal sheep lung DNA.


On the mechanism of neurotropism of vesicular stomatitis virus in newborn hamsters. Studies with temperature-sensitive mutants.

The virulence of temperature-sensitive mutants of vesicular stomatitis virus (VSV) injected subcutaneously into newborn hamsters was positively correlated with their tendency to generate revertants and with their leakiness in cultured hamster embryo fibroblasts maintained at 37 degrees C, the measured body temperature of the animals under our experimental conditions. The complementation group of the mutants seemed important only in that it tended to determine reversion frequency and leakiness. One non-reverting group I mutant (T1026), however, was much less virulent than would be expected from its extreme leakiness at body temperature. The disease produced by the less virulent mutants was characterized by neurological symptoms and led to delayed death, unlike the rapid deatth produced by virulent mutants. Infectious virus could be found in higher titres in the brains than in peripheral organs of such animals (with ratios as high as 10(8)). This neurotropism was not correlated with the complementation group of the mutant but was shown to be the consequence of survival for more than 3 days after injection. Age was not responsible for the effect. Animals injected at birth with T1026 were completely resistant to subcutaneous superinfection with the highly virulent wildtype virus HR at 3 to 4 days, though non-T1026-protected animals were completely sensitive. When HR was injected intracerebrally at 3 to 4 days, the T1026-protected animals allowed replication to high titres in the brain but not in peripheral organs, whereas non-T1026-protected animals allowed replication  to high titres in both brain and in peripheral organs. We suggest from these results that the observed neurotropism is produced by a resistance mechanism operative in peripheral organs but not in the brain; this resistance develops rapidly in newborn animals on exposure to virus and clears virus from the peripheral organs leaving it in the brain. It is possible that our effect represents a controlled and accelerated induction of the classical peripheral resistance of animals to various viruses which normally develops with age.


Inhibition of interferon action by vitamin A.

Simultaneous treatment of mouse cells with interferon and vitamin A (retinoic acid) resulted in an inhibition of interferon action. Increasing concentrations of calf serum decreased the inhibitory effect of retinoic acid on interferon action. Treatment of interferon with retinoic acid prior to the assay for interferon activity also resulted in a loss of interferon activity. Since the residual retinoic acid present after dilution of the interferon for assay was not sufficient to interfere with the assay, it is presumed that interferon and retinoic acid must interact in some fashion to inhibit interferon activity. Calf serum prevented the apparent interaction of retinoic acid and interferon. The loss of interferon activity which resulted from treatment of interferon with retinoic acid was dependent on temperature and time of incubation. Retinyl acetate (acetate ester of vitamin A) and retinal (vitamin A adehyde) only slightly inhibited interferon activity, while retinoic acid (vitamin A acid) and retinol (vitamin A alcohol) were similarly effective at inhibiting interferon activity. Another fat soluble vitamin, vitamin K1, did not inhibit interferon activity.


Absence of poly (A) from the infective RNA of Nodamura virus.

With the exception of phage Qbeta, the RNAs of all the other small icosahedral RNA viruses so far examined contain a poly (A) tract. This tract has been implicated in the infectivity of poliovirus RNA. We have now shown that Nodamura virus, a divided genome virus from which infective RNA can be extracted, does not contain any poly (A) tracts. This evidence with Nodamura virus shows that poly (A) is not a necessary requirement for the infectivity of virus RNA molecules.


DNA polymerase in pseudorabies virus infected cells.

The DNA polymerase activity in BHK 21/C13 cells infected with pseudorabies virus is inhibited by incubation with antiserum to pseudorabies but not by incubation with pre-immune serum or by antiserum to herpes simplex virus type 1 (HSV-1) or herpes simplex virus type 2 (HSV-2). It also differs from the cell enzyme and the enzymes in HSV-1 or HSV-2 infected cells in its requirement for KCl in the in vitro assay. It seems likely, therefore, that pseudorabies virus specifies its own DNA polymerase.


Anti-viral activity of single-stranded homopolynucleotides against encephalomyocarditis virus and Semliki Forest virus in adult mice without interferon induction.

Single administrations of poly C or poly I are anti-viral against infections of encephalomyocarditis (EMC) and Semliki Forest virus (SFV) in mice but poly U and poly A are not. The degree of protection is dose-dependent and mice which die do so at a later time when untreated controls even in a strain of mouse in which the time of death is not dependent on the dose of virus given. No circulating interferon is found after treating mice with poly C or poly I even at polynucleotide doses which give the same degree of protection as the interferon inducer, poly I:C. Several additional features distinguish the protection by poly C and poly I from interferon induction: the effect is low 24h before infection and maximal 6 h before infection, the effect is short-lived and mice do not show hypo-reactivation to repeated treatment. Limited treatment of mice with poly I:C, interferon or poly C before infection itself results in additional protection when poly C is also administered after infection, indicating that poly C has an effect after onset of virus replication. After infection poly C and poly I are both more effective by the intravenous route but before infection they are most effective when administered by the same route as the virus. Quantitative comparisons of the protective effects of poly C, poly I and the interferon inducer, poly I:C, are possible from dose response curves of the polynucleotides at different times relative to infection and by different routes of administration. The results are considered in relation to the presence of homopolyribonucleotide tracts in the viral genomes and effects on the reticulo-endothelial system of the mice.


Poliovirus proteins associated with the replication complex in infected cells.

Viral polypeptides associated with the membrane-free replication complex of poliovirus RNA were multiple in nature. The structural protein precursors [VP0, VP1, VP3] predominated, and because they were found in a cytoplasmic component with the same S value and density as the replication complex are likely to be attached to it in vivo. They were not present in the form of empty capsids. The electrophoretic polypeptide pattern of the membrane-bound replication complex was similar but showed a predominance of NCVPX or VP1, unless the cells were slightly depleted in amino acids when the non-structural polypeptide NCVP2 became important. Cystine was the only amino acid capable of reversing this depletion effect on its own.


[Poland's syndrome in a sterile woman].

In a woman aged 32, who came for consultation regarding her sterility, a case of Poland's syndrome was diagnosed in view of a brachysyndactylia and a homolateral agenesia of the large pectoral. The possible connexion between her sterility and this syndrome is discussed.


Studies on serotonin binding proteins of nerve ending membranes.

Synaptic membranes were isolated from rat brain homogenates by differential and density gradient centrifugation. Membrane proteins were solubilized by detergent buffer and assayed for serotonin-binding activity by adsorption of free 5-HT on charcoal. When the membrane extract was incubated with serotonin at +4 degrees C for various times, equilibrium was reached within 10 min. With increasing serotonin concentrations the specific part of binding was saturable whereas the non-specific part increased linear with the total 5-HT added. Kinetic analysis of the data revealed two different classes of binding sites with the apparent dissociation constants Kd1 = 5.3X10(-7) M and Kd2 = 1.1X10-5 M. The disociation reaction followed first order kinetics in two steps. The first step was very rapid, the second step proceeded with a half life time t1/2 of 16 min and a dissociation rate constant of k-1 = 7.2X10(-4) s-1. the binding was sensitive to heat and SH-blocking reagents and displaceable by serotonin in excess, d-LSD, and to a lower extent by 5-methoxytryptamine and tryptamine. The significance and localization of the binding sites at the membrane are discussed.


Comparison of the intermediary metabolism of fatty acids in denervated and dystrophic murine skeletal muscle.

Certain aspects of lipid metabolism have been examined in denervated muscle from normal mice and in dystrophic muscle from mice of the Bar Harbor strain 129. A number of parameters show no change or similar changes. For example, the utilization of palmitate-[1-14C] and palmitylcarnitine by mitochondria from denervated and dystrophic hind leg skeletal muscle showed parallel decreased in the oxidation of palmitate (30-42%) and palmitylcarnite (37-66%). A comparable study with acetylcarnitine showed a striking difference with no change evident in mitochondria from denervated muscle and 80-85% decrease in dystrophic muscle. The study of succinate dehydrogenase and the enzymes of beta-oxidation in the above mitochondrial preparation showed similar findings except for acyl CoA dehydrogenase activity (an enzyme with a regulatory role in beta-oxidation) which was significantly diminished (29%) in denervated muscle, whereas no change was observed in dystrophic muscle. The findings show a close parallel in a number of parameters but distinct differences were observed in denervated as compared with dystrophic muscle. It is unlikely that the muscular disorder in murine muscular dystrophy can be explained solely on the basis of denervation or the loss of a neural trophic factor.


Early onset myotonic dystrophy in association with polyneuropathy.

A patient with early onset of myotonic dystrophy, with associated neuropathy and epilepsy, is presented. It is postulated that his disorder was inherited through a recessive, pleomorphic gene. His differential diagnosis is discussed and the literature reviewed. The clinical variability of myotonic dystrophy is stressed and the diagnostic difficulties encountered in the younger age group.


Immunological differential diagnosis of human brain tumours.

Results of immunological experiments with various human brain tumours show differences between astrocytoma and glioblastoma multiforme when brain-specific alpha2-glycoprotein is used as an immunological marker. On this basis a tentative model of cytogenesis and oncogenic transformation of glial cells was presented. Morphological resemblances between the brain-specific alpha2-glycoprotein immuno-precipitation lines obtained from astrocytoma cells and from normal cerebral white matter treated with neuraminidase are discussed in relation to experimental and clinical studies of tumour cells treated with neuraminidase.


Hereditary demyelinating neuropathy in the trembler mouse.

The Trembler mouse suffers from a hereditary demyelinating neuropathy. Nerve conduction studies were performed on the sciatic nerve, median nerve and dorsal nerve of the tail. There was a prolonged terminal latency, and motor conduction velocities were less than 10 m/sec in all the nerves examined. The electrophysiological abnormalities correlate with the pathological findings of hypomyelination.


Neurological complication of antirabies vaccination in São Paulo, Brazil. Clinical and therapeutical aspects.

The author studied especially the clinical aspects and therapeutic results in 73 patients with neurological complication of anti-rabies vaccination. The neuroparalytic accidents and the most constant neurologic signs and symptoms were emphasized. The most common clinical syndrome was thoraco-lumbar meningomyelitis and there were CSF alterations in 88% of the cases studied. Eighteen patients presented neurologic complications after Fuenzalida vaccine: only 1 of them had Guillain-Barré syndrome, 1 had meningoradiculitis, 4 had myelitis and in the other 12 cases there was diffuse involvement of the nervous system especially of the spinal cord and meninges (meningomyelitis and meningoencephalomyelitis).


Retroperitoneal ganglioneuroblastoma: a kaleidoscope of neuronal degeneration. A light and electron microscopic study.

The light and electron microscopic features of an unique retroperitoneal ganglioneuroblastoma in a four-year-old female are described. The unprecedented concurrence of Hirano, zebra, membranous cytoplasmic (MCB), and Pick bodies in the same population of neoplastic, sympathetic ganglion cells provides further evidence for their non-specificity. Although the pathogenesis of the membranous cytoplasmic bodies in this tumor is unclear, they ostensibly arise within endoplasmic cisterns, similar to the proposed origin of membranous cytoplasmic bodies in Tay-Sachs disease. Both the apparent continuum between argyrophilic bodies and central chromatolysis, and the incorporation of various cytoplasmic constituents within neurofilamentous proliferations reflect some of the dynamic factors involved in the formation of Pick bodies.


Light-cored dense particles in mitochondria of a patient with skeletal muscle and myocardial disease.

A 15-year-old boy with skeletal muscle and myocardial disease was found to have large numbers of abnormal muscle mitochondria, the distinguishing feature of which was the presence of many light-cored dense particles. These particles bore a marked resemblance to those seen in situations where mitochondria accumulate calcium. If that is what they were, it remains to be determined whether such an accumulation of calcium, or other mitochondrial abnormality, played a role in the patient's muscle weakness. Alternatively, the granules might have represented evidence of virus involvement of the mitochondria, but this seems less likely.


The fine structure of astrocytes in the adult staggerer.

The fine structure of the cerebella of staggerer mice was studied. Particular attention was paid to the astrocytic processes which showed characteristic changes in the adult animal. These consisted of parallel configurations of slender sheet-like astrocytic processes frequently connected to one another by highly organized intercellular adhesive devices. The abnormal condition seen in staggerer is an exaggerated expression of the fundamental tendency of the astrocyte to form sheet-like peripheral processes.


Management of the uninhibited bladder by selective sacral neurectomy.

The authors report the treatment of incontinence due to uninhibited bladder contractions by selective sacral neurectomy in nine patients, four without evidence of neurological disease. A detailed and objective analysis of bladder and urethral function, together with quantitation of clinical features, was made before and after operation. Seven patients were either cured or greatly improved. The overall increase in bladder capacity and reduction of uninhibited activity were statistically significant. The resting urethral sphincter pressure was unchanged, but the contractility of the voluntary external sphincter was slightly impaired. Criteria for such neurectomies are discussed.


Controlled study of CCNU and radiation therapy in malignant astrocytoma.

The authors report 63 patients with biopsy-proved malignant (Grades 3 and 4) astrocytomas who were randomly placed in one of three treatment schedules within 2 weeks of surgery. One group (22 patients) received radiation therapy alone; the second group (22 patients) received 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea (CCNU) orally at intervals of 8 weeks; and the third group (19 patients) received combined radiation and drug therapy. Patients who received radiation therapy, with or without the drug, had a significantly longer survival than did those who received the drug alone. There was no difference in survival between the two groups who received radiation. The nitrosourea derivative CCNU does not seem to be an effective agent in the therapy of primary malignant brain tumors.


Increased localization of 99mTc-pyrophosphate in a bone island: case report.

A positive 99mTc-pyrophosphate bone scan is reported in a proven case of large compact bone island. Pyrophosphate uptake in this case is presumed to be due to either large size or growth of the bone island. A radionuclide bone scan does not always differentiate bone islands from metastatic or inflammatory sclerotic bone lesions.


Comparison of 18F and 99mTc-polyphosphate in orthopedic bone scintigraphy.

To compare 99mTc-polyphosphate and 18F for use in orthopedics, 79 patients were examined with both. Fifty cases were suitable for analysis. While the extraskeletal uptake of 18F was found to be negligible, 99mTc-polyphosphate may accumulate considerably in pathologic soft tissue, e.g., in soft-tissue tumors and in inflamed synovial tissue. This soft-tissue Tc accumulation may obscure the osseus uptake, notably in the examination of joint regions, commonly the regions of interest in orthopedics. After simultaneous administration of both agents, quantitative measurements were performed on specimens of bone and synovial tissue from diseased joints in human patients and in rabbits. The uptake of 99mTc-polyphosphate in synovial tissue was shown to be about seven times that of 18F, while their uptakes in bone were equal. In short, 99mTc-polyphosphate, a valuable tracer in general, is hardly the agent of choice in orthopedics.


Stoppage of glycogenesis and "over-shoot" of induction of lipogenesis and its related enzyme activities in the liver of fasted-refed rats.

To elucidate the causes of changes of carbohydrate metabolic pathways, the time course of utilization of dietary [U-14C]sucrose and induction of enzyme activities in the livers of rats were investigated. Adult male rats of BHE strain were refed after a fast of 2 days. The nutritionally complete refeeding diet contained 60% sucrose as the only source of carbohydrate. [U-14C]Sucrose was included in the diet on either day 1 or day 2, or both of refeeding. During the first day of refeeding, the radioactivity was incorporated mainly into liver glycogen which rose to over 100 mg/g. During the second day, little 14C appeared in the liver glycogen, which decreased sharply while glucose-6-phosphatase activity increased. The glycogenic pathway thus appeared to be blocked. On the other hand, 14C incorporation in the liver fat was minimal during the first day, but was quite extensive during the second day of refeeding. The enhanced lipogenesis was accompanied by large increases of activities of glucose-6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase and NADP-malic dehydrogenase. Results clearly indicate that the carbohydrate load in the liver of intact animals was initially metabolized by the glycogenic pathway. When glycogenesis stopped, carbohydrate was metabolized differently. The enhanced incorporation of [U-14C]sucrose into liver lipids indicates an increased formation of acetyl CoA and an accelerated formation and use of NADPH, probably from increasing dehydrogenase activities. Our data suggest that the blockage of synthesis of glycogen with the continuation of carbohydrate load was a primary cause in over-shooting induction of hepatic dehydrogenase activities and lipogenesis.


Gluconeogenic response to mannoheptulose in the rat.

Since administration of mannoheptulose induces temporary hyperglycemia, the present study was conducted to elucidate this phenomenon. The results indicate that mannoheptulose stimulates the activity of hepatic fructose-1,6-diphosphatase and phosphoenolpyruvate carboxykinase, and enhances incorporation of alanine into blood glucose and hepatic glycogen. In addition, mannoheptulose increases plasma levels of glucagon and hepatic cyclic AMP concentration. Gluconeogenic effects of mannoheptulose appear to be mediated by glucagon.