Purification of pre-prolactin mRNA from bovine anterior pituitary glands. Total cytoplasmic RNA, isolated from bovine anterior pituitary glands, directed the synthesis of primarily two proteins in a wheat germ cell-free system. Analysis of wheat germ cell-free products by sodium dodecyl sulfate-slab gel electrophoresis, direct immunoprecipitation with prolactin aatibody, and prolactin- and growth hormone-specific radioimmunoassay indicated that the two proteins were pre-prolactin and pre-growth hormone. Agarose-urea-gel electrophoresis of poly(A)-containing RNA revealed the presence of a large symmetrical RNA peak with an estimated size of approximately 15 S. The RNA contained in this peak was eluted from the gel and translated in the wheat germ cell-free system. Analysis of the translation products by electrophoresis, direct immunoprecipitation, and radioimmunoassay resulted in the detection of only pre-prolactin. The extent of pre-prolactin mRNA purification was assessed by synthesizing DNA complementary to gel-purified pre-prolactin mRNA and then following the hybridization of this cDNA to gel purified pre-prolactin mRNA. The result of this hybridization is consistent with the cell-free translation studies, namely that pre-prolactin mRNA is the predominant mRNA component of pituitary poly(A)-containing RNA.


Mucosal coenzyme A-dependent cholesterol esterification after intestinal perfusion of lipids in rats. Coenzyme A-dependent esterification of cholesterol was determined in intestinal mucosal homogenates prepared after duodenal perfusion of cholesterol-free lipid emulsions for 5 h in unanesthetized rats. Cholesterol esterification rates were lowest and the mucosal cholesterol pool was greatly reduced after the same lipid infusions that, in lymph fistula rats, had produced chylomicrons deficient in cholesterol esters. CoA-dependent esterification rates were sufficient to account for all the cholesterol esters secreted in mesenteric lymph chylomicrons. During triglyceride secretion by the gut, unesterified cholesterol for chylomicron membranes may be maintained both by suppressing mucosal CoA-dependent cholesterol ester formation and from a mobilizable unesterified cholesterol pool within the mucosa.


Characterization of a UGA-suppressing serine tRNA from Schizosaccharomyces pombe with the help of a new in vitro assay system for eukaryotic suppressor tRNAs. Two different allele-specific suppressor mutants of the fission yeast Schizosaccharomyces pombe produce opal (UGA) suppressor tRNAs. This was shown by the use of a new in vitro assay for eukaryotic nonsense suppression: a wheat germ extract is programmed with rabbit globin mRNAs and the readthrough products are studied. alpha-Globin is elongated upon addition of ochre (UAA) suppressor tRNAs, whereas beta-globin yields a readthrough product with opal suppressor tRNAs. This simple and very sensitive assay allowed the purification of the opal suppressor tRNA from S. pombe strain sup3-e. The pure tRNA can be aminoacylated with serine; thus, we conclude that this suppressor tRNA inserts serine in response to the UGA termination codon of pure rabbit beta-globin mRNA.


Synthesis and mode of action on axonal membranes of photoactivable derivatives of tetrodotoxin. Two photoactivable derivatives of tetrodotoxin have been synthesized. Electrophysiological experiments on crab giant axons and competitive binding with [3H]-tetrodotoxin for the tetrodotoxin receptor indicate that they are only 4.5 to 7.5 times less active than tetrodotoxin itself. These compounds give a reversible block of the sodium channel in the dark but after ultraviolet irradiation they provoke an irreversible blockade of the channel.


Malate dehydrogenase, inhibition of pig heart supernatant enzyme by iodoacetamide. Pig heart supernatant malate dehydrogenase is alkylated by 250 mM iodoacetamide at 37 degrees C in pH 7.5 Tris/acetate buffer which is 0.05 M with respect to acetate to form enzyme with 1,3-dicarboxamidomethyl histidine, 3-carboxamidomethyl histidine, 1-carboxamidomethyl histidine, carboxamidomethyl cysteine, and carboxamidomethyl methionine. 1,3-Dicarboxamidomethyl histidine forms with a stoichiometry of 1/enzyme subunit at a rate equaling the rate (approximately 1.24 +/- 0.09 X 10(-2) min-1) of enzyme inactivation by iodoacetamide. All other amino acid derivatives form at a slower rate initially or continuously. The enzyme is protected against inactivation by iodoacetamide through NADH binding, suggesting that there is an "essential" histidine residue at or near the active site.


Progesterone-induced secretory protein. NH2-Terminal sequence of pre-uteroglobin. Uteroglobin is a protein secreted by the rabbit uterus in response to progesterone. In cell-free translation systems, the mRNA for uteroglobin codes for a protein larger in size than the secreted protein. To investigate the relationship between these two forms, the NH2-terminal sequences of pre-uteroglobin and of uteroglobin have been determined. Uteroglobin was purified from rabbit uterine flushings and pre-uteroglobin was obtained by immunoprecipitation of the products of translation of poly(A)-rich endometrial RNA in the wheat germ system in the presence of single or multiple radioactive amino acids. Sequencing was performed by automated Edman degradation and the residue at each cycle was identified by chromatography. The larger size of pre-uteroglobin is accounted for by a 21-amino-acid leader sequence, containing 15 hydrophobic residues, at the NH2 terminus. The sequence Thr-Leu-Ala-Leu occurs twice in the leader region. In common with other secretory proteins, the transient hydrophobic extension at the NH2 terminus of pre-uteroglobin may function to assist transfer of the nascent peptide into the lumen of the endoplasmic reticulum, as proposed in the "signal" hypothesis.


Structural studies of the major high mannose oligosaccharide units from Chinese hamster ovary cell glycoproteins. The major high mannose-type glycopeptides present in Chinese hamster ovary cells have the compositions (Man)9(GlcNAc)2-Asn, (Man)8(GlcNAc)2-Asn, and (Man)6(GlcNAc)2-Asn. The structures of these glycopeptides were determined by the combination of methylation analysis, acetolysis, Smith periodate degradation, and alpha- and beta-mannosidase digestion. Their complete structures are: manalpha1 leads to 2Manaalpha1 leads to 6(Manalpha1 leads to 2Manalpha1 leads to 3)Manalpha1 leads to 6(Manalpha1 leads to 2Manalpha1 leads to 2Manalpha1 leads to 3)Manbeta1 leads to 4GlcNAcbeta1 leads to 4GlcNAc-Asn, Manalpha1 leads to 2Manalpha1 leads to 6(Manalpha1 leads to 3)Manalpha1 leads to 6(Manalpha1 leads to 2Manalpha1 leads to 2Manalpha1 leads to 3)Manbeta1 leads to 4GlcNAcbeta1 leads to 4GlcNAc-Asn, and Manalpha1 leads to 6(Manalpha1 leads to 3)Manalpha1 leads to 6(Manalpha1 leads to 2Manalpha1 leads to 3)Manbeta1 leads to 4GlcNAcbeta1 leads to 4GlcNAc-Asn. These structures are compared with the structures of the peptide-bound oligosaccharide intermediates that are processed to form complex-type oligosaccharides. From these results, it is proposed that the high mannose-type oligosaccharides are a product of "incomplete" processing of the protein-bound oligosaccharide along the same pathway which leads ultimately to the formation of a complex-type oligosaccharide.


Isolation of proteoglycan-hyaluronate complexes from bovine aorta. Two proteoglycan-hyaluronate complexes were extracted from bovine aorta with 4.0 M guanidinium chloride in the presence of protease inhibitors and purified by cesium chloride density gradient centrifugation under associative conditions. Complex I with higher buoyant density had 22% protein and 18% uronate, whereas Complex II had 11% protein and 23.6% uronate. Glycosaminoglycan (GAG) analysis of the complexes indicated that both complexes contained large amounts of hyaluronic acid, 13.6% and 24.0% of the total GAG in Complexes I and II, respectively. Chondroitin sulfates and dermatan sulfate constituted the remainder of the GAG. Chromatography on Sepharose CL 6B suggested that Complex I had an estimated molecular weight of 800,000 and Complex II, 250,000. The complexes were dissociated into proteoglycan and hyaluronate by cesium chloride density gradient centrifugation under dissociative conditions. The proteoglycans free of hyaluronic obtained from the complexes had similar protein and GAG composition. The molecular weight of the proteoglycan was estimated to be 80,000 and that of the hyaluronate of the complex, 90,000.


The preparation and properties of bovine enterokinase. Bovine enterokinase was purified from duodenal mucosa. The purification included an initial extraction with 2% deoxycholate, ammonium sulfate fractionations, DEAE-cellulose chromatography, and affinity chromatography on basic pancreatic trypsin inhibitor (Kunitz) (PTI)-Sepharose. The purified enzyme contained 35% carbohydrate; it had a molecular weight of 150,000, with a heavy (115,000) and light (35,000) chain connected by one or more disulfide bonds. Enterokinase hydrolyzed lysine and arginine substrates and slowly reacted with the trypsin active site titrant 4-methylumbelliferyl-p-guanidinobenzoate. The enzyme activated bovine trypsinogen with kinetic parameters similar to those of other preparations of enterokinase. Bovine enterokinase was inhibited by Kunitz pancreatic trypsin inhibitor with a Kassoc of 2 X 10(8) M-1 and only weakly by other proteinase inhibitors. The amino acid composition differed from bovine enterokinase isolated from duodenal contents (Anderson, L.E., Walsh, K.A., and Neurath, H. (1977) Biochemistry 16, 3354-3360). The mucosal enzyme and the duodenal contents enzymes also differed in the size of the heavy and light chains. The mucosal enterokinase more closely resembled the properties of porcine enterokinase (Baratti, J., Maroux, S., Louvard, D., and Desnuelle, P. (1973) Biochim. Biophys. Acta 315, 147-161). The amino acid composition and size of the light chain were also similar to bovine trypsin.


Identification of ATP citrate lyase as a phosphoprotein. ATP citrate lyase has been purified from rat liver by a new procedure which results in high yields of an intact and stable enzyme. The pure lyase (specific activity approximately equal to 10 at 25 degrees C) exhibits a single protein band upon sodium dodecyl sulfate (SDS)-gel electrophoresis (Mr = 110,000). This procedure minimizes protease degradation that usually occurs when the enzyme is isolated by previously described isolation methods. In addition, the lyase is shown to be a phosphoprotein. 32P-labeled lyase has been purified from rat liver following an intraperitoneal injection of inorganic [32P]phosphate into the animals. It has been demonstrated that this phosphate (structural phosphate) behaves as a serine phosphate and is not the same as the enzyme-bound phosphate (catalytic phosphate) that is derived from ATP during the lyase reaction. There are 2 structural phosphate residues for each enzyme tetramer molecule. Removal of the structural phosphate has been accomplished using a partially purified phosphatase derived from rat liver. The dephospholyase has the same Vmax as the native phosphoenzyme. Evidence indicates that the structural phosphate resides in a protease-sensitive region of the native enzyme.


An electron microscopic study of pyruvate carboxylase. Pyruvate carboxylases, purified to homogeneity from sheep and chicken liver, have been examined in the electron microscope and found to have a splayed tetrahedral structure which, in some views, appears similar to that described for this enzyme isolated from yeast. The square-planar tetrameric image previously ascribed to pyruvate carboxylases from vertebrate sources was shown to be that of a molecule which is not only larger than pyruvate carboxylase, but also devoid of biotin.


A re-examination of the electron microscopic appearance of pyruvate carboxylase from chicken liver. Electron microscopic studies of chicken liver pyruvate carboxylase conducted under a variety of conditions show that this enzyme has an overall rhombic appearance and is comprised of four nonspherical subunits. The square planar tetramers originally identified by Valentine et al. (Valentine, R.C., Wrigley, N.G., Scrutton, M.C., Irias, J.J., and Utter, M.F. (1966) Biochemistry 5, 3111-3116) as pyruvate carboxylase have been shown to represent a minor protein contaminant found in many impure preparations of this enzyme. The contaminating protein has been separated from pyruvate carboxylase and further purified. It does not contain biotin and its constituent polypeptides are smaller than those of pyruvate carboxylase. This protein, whose function is not yet identified, shows a strong tendency to aggregate and is highly visible under many conditions of electron microscopy. Several lines of evidence support the thesis that the nonsquare tetramers are pyruvate carboxylase. When essentially homogeneous material is examined with a variety of different negative stains, numbers of intact molecules represent 20 to 70% of the visible protein. These tetramers, like pyruvate carboxylase, are very cold-labile and are protected from dissociation under these conditions by acetyl-CoA, a specific activator of this enzyme. Also, the structures form complexes with avidin and antibiotin antibody and thus, like pyruvate carboxylase, contain biotin.


Rate of potassium-sodium exchange by human lymphocytes: prediction of the cooperative adsorption model. The equilibrium parameters of potassium-sodium distribution in human lymphocytes, determined experimentally in the preceding study (Negendank and Shaller, '79), were incorporated into a stochastic treatment of the cooperative adsorption model in order to predict the kinetics of "active" potassium-sodium exchange. The rate of uptake of potassium, in potassium-depleted, sodium-loaded cells, is complex and deviates markedly from simple exponential functions. The sigmoid form of the exchange data closely followed the predicted curve. This result enhances one's confidence in the usefulness and applicability of the cooperative adsorption model, and adds further support to the association-induction hypothesis as a coherent theory of cell physiology.


The effects of dopamine on prostaglandin E1-elicited electrophysiological changes in a neuronal somatic cell hybrid. PGE1 elicited a slow, dose-dependent membrane depolarization with an increase in membrane conductance in the somatic cell hybrid TCX11. The ED50 was 1-2 X 10(-8) M with maximal responses at 1-5 X 10(-7) M. Dopamine (DA) reversed the effect of PGE1 and caused the membrane potential and resistance to return to control levels. Chronic exposure of cells (measured in minutes) to DA alone would not cause this hyperpolarization. 5-HT was also tested and failed to consistently reverse the PGE1 effects. Chlorpromazine antagonized the effects of DA on the PGE1 response. The electrophysiological results reported here using TCX11 cells are discussed in light of previously reported biochemical results describing interactions of PGE1 and DA, and the electrophysiological effects of DA alone.


Binding of Salmonella minnesota R-form glycolipid mR595 to rat fibroblasts and its effect on cell metabolism and cell behaviour. Trypsinized normal rat embryo fibroblasts and untrypsinized and trypsinized transformed rat fibroblasts have two orders of binding sites for bacterial glycolipid mR595. The high order sites fix 1--3 micrograms glycolipid mR595/10(5) cells and those of the low order fix about 6 microgram glycolipids mR595/10(6) cells. Ca++ is required for the low order glycolipid mR595 binding to be trypsinized but not to the untrypsinized transformed rat fibroblasts. The low order binding is temperature dependent with the transition temperature lying between 25 and 37 degrees C. Exogenously added ganglioside and glycoproteins contained in the fetal calf serum do not inhibit fixation of glycolipid mR595. Only beta-lipoprotein at high concentrations is slightly inhibitory. Glycolipid mR595 fixation to transformed fibroblast does not alter their morphology and appears to slightly improve cell attachment to substratum. Glycolipid mR595 fixation results in a lengthening of the S-phase of the cell cycle and a reduction in 2-deoxyglucose uptake. Uptake of inorganic phosphate is not affected. Inhibition of phospholipid synthesis is observed in mR595 fixed fibroblasts whereas synthesis of cell surface glycoproteins and the content of cellular gangliosides is not affected.


Lymphocyte response to phytohemagglutinin: intracellular volume and intracellular [K+]. The effect of phytohemagglutinin (PHA) on lymphocytes was examined with respect to free intracellular water volume and intracellular [K+]. At a cell concentration of 30 X 10(6) lymphocytes/ml in modified Hank's Buffered Salt Solution (HBSS) in the presence of 10% human AB serum, addition of PHA at 3 mg/ml resulted in a 24-27% decrease in free intracellular water space within 30 to 60 minutes and a return to control level after three hours. A larger change in intracellular water (44%) was observed under similar conditions in the absence of serum. The absolute intracellular K+ content did not change after PHA addition, but the cell water volume decrease arising from PHA addition resulted in a 29% increase in intracellular [K+] at 60 minutes. The decrease in lymphocyte water volume induced by PHA was also observed for concanavalin A which stimulates lymphocyte proliferation, but not for wheat germ lectin, an agglutinating agent which is not mitogenic. Thus, volume regulation may be closely associated with the mitogenicity of these compounds.


Studies on intercellular adhesion. III. Adhesion of cells to isotypic or heterotypic collecting particles and monolayers. Adhesion between cells of different tissues from chick embryos was studied by the modified collecting particle (Pessac et al., '77a) and stationary monolayer assays. These assays measure the number of 3H labeled cells that adhere to the surface of cell aggregates and tissue fragments or on top of cell monolayers. The numbers of cells from a given suspension that adhere to identical or different cells can be compared. Intercellular adhesion may be considered as tissue specific if, in identical conditions, more cells adhere to identical cells than to cells from different tissues. Neural retina, cerebrum, optic tectum, liver and kidney cells from 9-day-old chick embryos showed no tissue specificity of adhesion, while heart cells adhered preferentially to collecting heart fragments.


Isolation and cell cycle analysis of temperature-sensitive mutants from Chinese hamster cells. Mutants temperature-sensitive for growth have been isolated from the established line of Chinese hamster fibroblasts Wg1A. These mutants, together with the ones previously isolated by Roscoe et al. ('73), have been characterized with regard to their cell cycle properties. Most of them become arrested in the G1 phase of the cell cycle when incubated under restrictive conditions. By performing temperature shift experiments with synchronous cultures, the execution steps of most of the mutated functions have been located within the second half of the G1 phase.


Establishment of a rat hepatoma cell line which has ornithine carbamoyltransferase activity and grows continuously in arginine-deprived medium. The epithelial cell line, H4-II-E derived from Reuber hepatoma H35 has no significant activity of ornithine carbamoyltransferase (OCT, EC 2.1.3.3) and is not able to grow in arginine-deprived medium. A multi-step selection procedure is described which selects from HR-II-E populations, cells with OCT activity which can grow in arginine-deficient, ornithine-supplemented media.


The initiation and elongation steps in protein synthesis: relative rates in Chinese hamster ovary cells during and after hyperthermic and hypothermic shocks. The relative rates of the initiation and elongation phases of protein synthesis have been determined in heat- and cold-shocked CHO cells from measurements of the incorporation of 35S-methionine into N-terminal and internal positions of growing peptides by a modified Edman degradation. When the cells are shifted from 37 degrees C to temperatures between 10 degrees C and 34 degrees C, the rate of initiation is at first reduced more extensively than that of elongation. After 20 to 30 minutes at the lower temperature, however, the cells undergo a metabolic adjustment which includes increasing the rate of initiation until it corresponds to the rate of elongation at that temperature. Calculated apparent energies of activation for initiation and elongation are in reasonable agreement with those determined in other mammalian cells. When the cooled cells are returned to 37 degrees C, the rates of initiation and elongation recover immediately but do not exceed the control values. Exposure to elevated temperatures (43 degrees C) causes an immediate cessation of initiation and thus a delayed inhibition of elongation; upon return to 37 degrees C, the rate of initiation is transiently elevated above the control rate, and the rate of elongation returns to the control rate after a 2- to 3-minute delay. Hence, a factor which leads to supranormal rates of initiation may accumulate at high but not at low temperatures.


Patterns of peptide synthesis in senescent and presenescent human fibroblasts. Peptide production in senescent and presenescent human foreskin fibroblasts was measured using 2-dimensional polyacrylamide gel electrophoresis. This procedure permits the visualization of a cohort of the major peptides being produced. Among this cohort of over 500 peptides only two were found to differ in relative amount in that more was being produced in senescent cells. This difference was confirmed by measurements of the relative intensity of the peptide spot. This difference was senescent cell-specific and not due to the differences in rate of growth of senescent and non-senescent cells.


Elevation of HeLa cell 3-hydroxy-3-methylglutaryl coenzyme A reductase activity by glucocorticoids: possible relationship to the cell cycle. Elevation of 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMG-CoA reductase, EC 1.1.1.34) activity by glucocorticoids was shown to be dependent on the concentration of hormone in the medium over a range of 5 X 10(-10) to 1 X 10(-8) M, although the presence of steroid in the assay at 10(-5) M elicited no increase in activity. There was a demonstrated time dependence for the addition of dexamethasone i.e., from zero to six hours after serum removal, addition of hormone resulted in the same peak acitvity; addition at 12 hours gave slight elevation but resulted in an extended maintenance of the peak level of activity; addition at 24 hours showed no effect. When cycloheximide was added to the above times, subsequent kinetics showed identical decay of the enzyme activities from control and treated cultures at 6 and 24 hours, but at 12 hours the activity from dexamethasone treated cells exhibited an extended lag before the onset of decay, which then proceeded at the same rate as the control. The continuous presence of the hormone was not necessary for the induction to continue and the addition of Actinomycin D to cultures incubated in the presence of hormone resulted in an immediate decay of catalytic activity without evidence of "superinduction." The addition of progesterone at the same time as dexamethasone resulted in a concentration-dependent inhibition of the augmentation, suggesting the involvement of the glucocorticoid receptor in the elevation of HMG-CoA reductase activity. Flow microfluorometric (FMF) analysis of hormone treated cells indicated a delayed entrance into the DNA synthesis (S) phase of the cell cycle. The temporal relationships between this cell cycle perturbation and HMG-CoA reductase elevation are discussed.


Metabolic correction of fucosidosis fibroblasts by human alpha-L-fucosidase. Human alpha-L-fucosidase, purified from placenta, was taken up from the culture medium by skin fibroblasts from patients with fucosidosis (alpha-L-fucosidase deficiency). The rate of uptake was low (uptake coefficient = 6 X 10(-4) ml.mg-1.h-1). Intracellular alpha-L-fucosidase activity was directly proportional to enzyme in the medium up to an activity of at least 40 nmoles/min/ml. No evidence for saturation of specific cell-surface receptors was seen. However, uptake was reduced by 75% by 1 mM mannose-6-phosphate and by 50% by 1mM glucose-6-phosphate, suggesting that uptake may be mediated by a receptor recognising a phosphorylated sugar or an analagous compound. Enzyme taken up by the cells was most active in subcellular fractions enriched with lysosomes and had an isozyme pattern, by isoelectric focusing, identical to that of the original enzyme preparation. Fucosidosis fibroblasts were shown to accumulate low molecular-weight, fucose-containing compounds to a level several times greater than control cells. This stored material was eluted from Sephadex G-25 as an asymmetrical peak with an elution volume of approximately twice the void volume of the column. Addition of placental alpha-L-fucosidase to the culture medium of fucosidosis fibroblasts prevented excessive accumulation of fucose-containing material and accelerated the breakdown of material accumulated prior to enzyme uptake.


The accumulation of manganese from the environmental medium by the egg of Oryzias latipes. The manganese content of the egg and embryo of the Medaka, Oryzias latipes was determined by activation analysis. A remarkable increase in the amount of manganese in the egg was observed within one hour after fertilization. The rate of increase was reduced by the gastrula stage and the concentration of manganese remained unchanged at a later stage. The accumulation of manganese by the Oryzias egg was discussed in relation to the effect of manganese on respiratory enzyme systems.


Cell cycle control by Ca++-ions in mouse 3T3 cells and in transformed 3T3 cells. Total cellular calcium levels do not change when 3T3-4a cells stop proliferating due to serum depletion, or when serum-arrested quiescent cells are incubated for up to 44 hours in calcium-deficient medium (approximately 10 micrometer Ca++). Upon stimulation with dialyzed serum cells enter S and progress through at least one cycle even at extremely low calcium levels in the culture medium (greater than or equal to 10 micrometer). Cells divide until a final cell density is attained which is proportional to the calcium concentration in the medium and cells reversibly arrest in G1. Cells which arrested in G1 in medium containing less than or equal to 26 micrometer Ca++ in the presence of excess serum can be stimulated to enter S in response to added calcium after a prereplicative phase of 14 to 16 hours. Serum does not affect 45Ca-uptake in these cells. Benzo[a]pyrene transformed 3T3 (BP3T3) cells have a 100--200 times lower Ca++-requirement than 3T3 cells but arrest in G1 at low Ca++ levels. In contrast, SV40-virus transformed 3T3 (SV3T3) cells that grow without restriction in monolayer cultures have even lower Ca++-requirements for growth than BP3T3 cells and have no Ca++-sensitive restriction point. Therefore, 3T3 and BP3T3 cells have retained the capacity to sense intracellular Ca++-pool sizes and to arrest in G1 at subthreshold cellular Ca++-levels.


Growth and electron microscopic studies on an experimentally established bacterial endosymbiosis in amoebae. A strain of nonsymbiotic A. proteus was infected with endosymbiotic bacteria isolated from another strain of amoeba which had become dependent on the symbionts after a few years of spontaneously established symbiosis. In the newly infected amoebae, the bacteria avoided digestion and multiplied at a faster rate than the hosts, reaching the maximum carrying number (about 42,000 per amoeba) in fewer than ten cell generations of the hosts. The experimentally infected amoebae were also examined under the electron microscope, and the development of bacteria-containing vesicles was followed. The results show that the infective bacteria that were initially harmful to host amoebae have become harmless and that they have changed in their mode of multiplication during the course of establishing a stable symbiosis with their hosts.


Basis for differential cellular sensitivity to 8-azaguanine and 6-thioguanine. Cellular resistance to the cytotoxic purine analogues 8-azaguanine (AG) and 6-thioguanine (TG) is usually mediated by a mutation leading to the loss or reduction in hypoxanthine phosphoribosyltransferase (HPRT) activity. However, stable AG-resistant variants have often been shown to contain wild-type levels of HPRT, while cellular resistance to TG is always accompanied by a profound deficiency in HPRT activity. Such AG-resistant, HPRT-positive cells are still sensitive to TG. To investigate the basis of this differential sensitivity, we examined the inhibition of the HPRT activity by AG and TG in whole cells, in cell-free extracts, and with purified mouse HPRT. In addition, the relative incorporation and utilization of AG and TG by L929 cells were determined under a variety of culture conditions. Results show that, compared to TG, AG is generally a very poor substrate for HPRT. Incorporation of radioactive AG by HPRT-positive cells was extremely sensitive to the free purine concentrations in the medium, so that under the usual culture conditions employing undialyzed serum, cellular uptake and utilization was minimal even when relatively high levels of AG were present. In contrast, the incorporation of radioactive TG was comparable to that of a natural substrate, hypoxanthine. The results indicate that the differential cellular sensitivity to AG and TG is due to the difference between these two guanine analogues as substrates of HPRT. Additional data indicate also that cellular resistance to TG is mediated exclusively by HPRT deficiency, but resistance to very high levels of AG may result through at least two other mechanisms not involving HPRT deficiency. These observations may help resolve some of the conflicting data in the literature, and demonstrate that TG is a better selective agent for the HPRT-deficient phenotype.


Effect of amino acid deprivation on DNA synthesis in BHK-21/C13 cells. Removal of serum from BHK-21/C13 cells in culture results in a decline in thymidine incorporation extending over five days. Additional removal of any of several amino acids results in a rapid decrease in incorporation of thymidine to negligible levels by 24 hours. Replacement by complete medium then provokes a synchronous wave of DNA synthesis after only ten hours with DNA synthesis first increased at six hours. Starvation for glutamine results in a rapid decline in protein synthesis over the 24 hour period when DNA synthesis is falling. However, there is considerable degradation of total protein during this period, and RNA degradation is also greatly increased. Concurrently, synthesis of RNA falls to less than 10% of that in control cells.


Potassium-sodium distribution in human lymphocytes: description by the association-induction hypothesis. Human lymphocytes were equilibrated for 48 hours over a wide range of external potassium levels, and their contents of potassium, sodium, and water determined. As external potassium rose from zero, cell potassium rose steeply in a sigmoidal fashion, reached half-saturation at 0.4 mM esternal potassium, and then saturated at 129 mmoles/kg cells. The saturable cell potassium exchanged mole-for-mole with sodium. Analysis of the saturable components by a statistical-mechanical adsorption model demonstrated a cooperative intraction between sites determining equilibrium potassium-sodium distribution. Superimposed upon the saturable fraction of cell potassium was a smaller one that was non-saturable with increasing external potassium to at least 64 mM, and that, when expressed as mmoles/liter cell water, existed in a ratio to external potassium of 0.6. The results strongly support the association-induction hypothesis, which predicts a small non-saturable component of ions determined by exclusion from oriented cell water and a cooperative interaction between sites throughout the cell that associate with potassium or sodium.


Determination of propranolol and six metabolites in human urine by high-pressure liquid chromatography. A method for the determination of propranolol and six of its metabolites, as well as their glucuronide and/or aryl sulfate conjugates in human urine is described. Propranolol and its basic and neutral metabolites are extracted into ether at pH 9.8, evaporated to dryness, reconstituted, separated on a reversed-phase, high-pressure liquid chromatographic system and quantitated using fluorescence detection. The aqueous urine aliquot is then made acidic and the acid metabolites extracted and measured using similar methods. The presence of 2% sodium metabisulfite in all urines collected is essential to ensure the stability of 4-hydroxy-propranolol during collection and storage. Preliminary data is presented from 24-h urine samples collected from three patients chronically receiving propranolol.


Determination of quinidine and its major metabolites by high-performance liquid chromatography. A specific and precise assay, capable of quantitating in human plasma simultaneously but separately quinidine, dihydroquinidine and the quinidine metabolites 2'-quinidinone, 3-OH-quinidine and a third metabolite found--tentatively identified as the product formed by rearrangement of quinidine-N-oxide-is reported. The assay uses a normal phase high-performance liquid chromatographic (HPLC) system with a variable-wavelength UV detector at 235 nm and has a limit of sensitivity at approximately 20 ng/ml. The mobile phase consists of hexanes-ethanol-ethanolamine (91.5:8.47:0.03). A 2-ml plasma sample is worked up by adding primaquine base as an internal standard and extracting with ether-dichlormethane-isopropanol (6:4:1). The organic extract is evaporated and the residue reconstituted in 100-600 micron1 of mobile phase and an aliquot injected onto the column. Comparison of this procedure with the Edgar and Sokolow (dichloroethane) extraction--fluorescence procedure and with the Cramer and Isaksson (benzene) double extraction--fluorescence assay indicates that both fluorescence procedures give quinidine concentrations up to 2.3 times those determined by HPLC. These discrepancies were shown to be due to carry-over of metabolites and some extraneous background fluorescence.


Gas-liquid chromatography of isobutyl ester, N(O)-heptafluorobutyrate derivatives of amino acids on a glass capillary column for quantitative separation in clinical biology. A gas chromatographic method adapted to routine analysis has been developed for quantitative separation on glass capillary columns for free proteic and other known amino acids normally or abnormally found in physiological fluids. The procedure involves ion-exchange chromatography and isobutyl ester, N(O)-heptafluorobutyrate derivatization of free plasma and urine amino acid samples. Derivatized components were ascertained by combined gas chromatography-mass spectrometry. The use of glass for the capillary column is mandatory to achieve qualitative and quantitative analysis of the known occurring amino acids in urine and small plasma samples. Quantitative analysis of several types of human amino acid disorders are presented.


Surfactant ion-pair high-performance liquid chromatography of tryptophan and some of its metabolites in biological fluids. A rapid, sensitive assay for tryptophan and some of its metabolites in urine, plasma and saliva has been developed using sodium dodecylsulphate as a pairing ion in a surfactant ion-pair high-performance liquid chromatography technique. The method is highly selective for tryptophan which is separated from its main indoleamine metabolites, 5-hydroxytryptophan, 5-hydroxytryptamine and 5-hydroxyindoleacetic acid, and from kynurenine. The usefulness of the assay has been demonstrated in plasma level and urinary excretion studies of orally administered tryptophan.


Ways of collagen separation in pathologically altered tissue. A system of chromatographic methods using two successive DEAE-cellulose chromatographic steps and two successive separations on Bio-Gel A-1.5 m has been worked out for the separation of individual collagen types. The success of the procedure is based on the preliminary removal of proteoglycans during the first DEAE-cellulose run. Alternatively it is possible to replace chromatographic steps, following the removal of proteoglycans, with fraction precipitation.


Longitudinal urinary trace amine excretion in a human male. The urinary excretion of a beta-phenylethylamine (PE), m-tyramine (mTA), p-tyramine (pTA) and tryptamine (TR) in their unconjugated (free) and conjugated (except tryptamine) forms, was examined in a male human subject over a total period of 28 days. The average excretion values were (in microgram per 24 h, mean +/- standard error): PE, free 3.19 +/- 0.21, conjugated 6.85 +/- 0.52, mTA, free 98.0 +/- 2.2, conjugated 106.1 +/- 18.6; pTA, free 427 +/- 12, conjugated 571 +/- 142; and TR, free 79.4 +/- 2.8. This data, when considered along with other published information, permits the suggestion that probably mTA and pTA in the unconjugated form are exclusively formed endogenously; whether or not this also pertains to PE and TR is less clear. In all cases, the conjugated amines derive from both exogenous and endogenous sources.


Spectrum of total fatty acids in cerebrospinal fluid determined by gas chromatography. A simple gas-liquid chromatographic method for the determination of the spectrum of fatty acids in a small volume of cerebrospinal fluid (CSF) is presented. In a group of 49 neurological patients it has been found that in the CSF of the controls (n = 12) there are the following main fatty acids: oleic (27.28%), palmitic (23.23%), stearic (12.21%), linoleic (7.66%), myristic (5.02%), and palmitoleic (4.51%). Altogether 28 fatty acid methyl esters (FAME'S) from 12:0 to 22:2 have been tentatively identified. The majority of them appeared irregularly, sometimes in less than in 10% of cases. The composition of FAME'S in the CSF of patients with lumbar discopathy and with acute ischaemic cerebrovascular accidents does not differ from the control group. A significant difference (P less than 0.01) has been found in the group of hypophyseal adenomas in which the amounts of practically all saturated FAME'S with an odd carbon number are elevated. The same applies to the 18:0 and 20:0 compounds. In the group of atrophic and degenerative CNS processes the palmitic and stearic acids predominated to the detriment of oleic and linoleic acids.


A specific and sensitive method for the determination of the anticoagulant phenprocoumon in plasma. A specific thin-layer chromatographic assay for phenprocoumon has been developed with a sensitivity of 5 ng/ml of plasma, using only 0.2 ml. This sensitivity is more than 20 times higher than that of the published methods. The drug is extracted from acidified plasma, an aliquot of the extract is applied to a silica-gel thin-layer plate and separated from interfering substances. The quantity of phenprocoumon is determined by fluorescence densitometry in situ. The standard deviation of the whole procedure is less than +/- 3%. The new procedure permits pharmacokinetic studies with low doses of phenprocoumon to be performed on volunteers. Furthermore, due to the high sensitivity of the method, it is possible to determine the free drug fraction of this highly protein-bound substance in the plasma of patients. It was shown that, in the therapeutic concentration range, phenprocoumon is bound by about 99.5% to the plasma proteins. Since the assay is simple and quick to perform, a large series of plasma samples can be analysed without any problems.


Caffeine determination in rat plasma. A comparative study of micromethods. Confronted with the need for a sensitive (1 mg/1), specific and reproducible (less than 5%) method for the determination of caffeine in small plasma samples (10-50 microliter) of small laboratory animals, two existing methods, radioactive labelling and gas chromatography, were adapted. The first step, common to both methods, is chloroform extraction, followed by either gas-chromatographic analysis or radioactivity measurement. These methods were compared by using a common internal standard, labelled caffeine, measured before and after the extraction step. The initial requirements were fulfilled and the correlation of the results proved to be excellent. The methods can be extended to animals other than the rat, and to organs or biological fluids other than plasma. The very small amount of plasma needed for the determination allows pharmacokinetic studies to be conducted in small laboratory animals. Further, by combining the two methods it is possible to perform rather complex investigations, such as evaluating the extent of caffeine metabolism and, at the same time, determining levels in the case of chromic administration.


Extractive alkylation of sulphonamide diuretics and their determination by electron-capture gas chromatography. The extractive alkylation of 11 sulphonamide diuretics has been evaluated using tetrabutylammonium, tetrapentylammonium or tetrahexylammonium as counter ion at different pH values and methyl iodide in methylene chloride as the organic phase. The sulphonamides are methylated within 20 min with tetrahexylammonium as counter ion in 0.2 M sodium hydroxide at 50 degrees. The derivatives have been identified by mass spectral and nuclear magnetic resonance analysis. Relative retentions of the derivatives are given using 1% SE-30 as the stationary phase. A contaminant, dimethylsulphuric acid, occurs in methyl iodide and seriously disturbs the gas chromatographic analysis. The application of the extrative alkylation to biological samples is demonstrated by the direct analysis of acetazolamide in serum. 0.1 M tetrapentylammonium in 0.5 M sodium hydroxide is suitable as the aqueous phase with 1.6 M methyl iodide as alkylating reagent in methylene chloride. The trimethyl derivative of acetazolamide formed has been determined by electron-capture gas chromatography down to 0.5 microgram/ml in a 0.1-ml serum sample. The relative standard deviation at the 10 microgram/ml level is 6.6% (n = 10).


Separation of geometric isomers of retinyl ester, retinal and retinol, pertaining to the visual cycle, by high-performance liquid chromatography. A method for the analytical and/or preparative separation of the 13-cis, 11-cis, 9-cis and all all-trans isomers of retinyl palmitate ester, retinal and retinol by high-performance liquid chromatography is described. A straight-phase adsorption system consisting of Si 60 silica gel as the stationary phase and mixtures of n-hexane and dioxane as mobile phases were employed, using photometric detection at either 320 or 360 nm, depending on the nature of the compounds being studied. With simple adaptation of the phase system, all geometric isomers within each group could be separated. The precision of the method was 0.5% at the 10-microgram (about 40-nmole) level and 4% at the 10-ng (about 40-pmole) level (n = 3). The detection limit was about 0.5 ng (about 2 pmole). The suitability of the phase system is demonstrated by chromatograms of test mixtures and of eye extracts. The fractionation of 0.5 mg of an isomeric sample could be achieved on a column of length 250 mm and I.D. 10 mm.


Affinity chromatography at sub-zero temperatures. A model study with porcine pancreatic elastase. A new variety of affinity chromatography of enzymes is described which consists of building up an affinity adsorbent composed of a real substrate. The chromatography is performed at a sub-zero temperature where the turnover of the enzyme is very low or stopped. As a model system Sepharose-bound L-trialanine p-nitroanilide was for used the affinity binding of porcine pancreatic elastase, which was adsorbed to the column in a hypersaline medium at--14 degrees and eluted from the column at the same temperature using 50% (v/v) ethylene glycol. The affinity adsorbent proved to be vary specific as it did not retain trypsin, chymotrypsin and ovalbumin and retained only 20% of cytochrome c.


Comparison of elution patterns of proteins chromatographed on controlled-pore glass and carboxymethyl-cellulose. As controlled-pore glass (CPG) has anionic silanol groups, we compared the elution patterns of proteins chromatographed on CPG with those on carboxymethyl (CM)-cellulose using standard proteins and two protein mixtures, rabbit serum and bovine parotid extracts. The results showed that the order of proteins eluted from CPG was similar to that from CM-cellulose, although some differences were found. The adsorption of proteins on CPG was slightly stronger than that on CM-cellulose and the separation of proteins on CPG was slightly better. Some conditions, such as amounts of proteins loaded on the column, pH, temperature and kind of buffer solution for elution, were investigated. The pH of buffer solutions was important in the adsorption chromatography of proteins on CPG and the useful pH range was 7.0-8.5.


High-performance liquid chromatographic analyses of sulbenicillin and carbenicillin in human urine. A high-performance liquid chromatographic method has been developed for the determination of sulbenicillin (SBPC) and carbenicillin (CBPC) excreted in human urine. The method uses reversed-phase ion-pair chromatography with a bonded hydrophobic stationary phase and methanol-tetra-n-butylammonium bromide solution as the mobile phase. Urine is filtered through a micropore membrane and the filtrate introduced directly into a liquid chromatograph. An in vivo experiment was conducted by administering intravenously 1 g of SBPC or CBPC to volunteers and analysing their urine. One-compartment model analysis of the time-course data revealed that 73.1% of the amount of SBPC dosed was excreted in the urine with a rate constant of 0.579 h-1, and 91.5% of CBPC with a rate constant of 0.845 h-1.


Qualitative and quantitative determination of metapramine and its metabolites in biological materials. The qualitative and quantitative determination of metapramine and its metabolites, after selective solvent extraction from biological materials, are achieved by gas-liquid chromatography with or without an internal standard, carried out directly or after derivatization, spectrofluorimetry and one- or two-dimensional thin-layer chromatography. The gas chromatographic procedures are the most sensitive for the quantitative determination fo the unchanged compound: 15 and 35 ng/ml in plasma and 5 and 10 ng/ml in urine; the detection limit for thin-layer chromatography is about 100 ng. These techniques can be used for pharmacokinetic studies and therapeutic controls, and also in analytical toxicology.


Simultaneous determination of endogenous norepinephrine and dopamine-beta-hydroxylase activity in biological materials by chemical ionization mass fragmentography. A new clean-up procedure is described for the quantitation of biogenic amines and their metabolites by gas chromatography-chemical ionization mass fragmentography. The procedure is carried out in an acidic, non-aqueous medium. The metabolites as well as the amines are extracted with greater than 92% recovery. Using a CPG-10 column, the procedure is most suitable for the purification of these amines and their metabolites from the extract. It has been applied to the simultaneous determination of endogenous norepinephrine and dopamine-beta-hydroxylase (DBH) activity in rat adrenal by use of [2H2]dopamine as a substrate and [2H6]norepinephrine as an internal standard. The quantitation limit of the DBH activity is 0.1 pmole/mg of rat adrenal per hour.


Separation of unconjugated pteridines by high-pressure cation-exchange liquid chromatography. In the course of determining the levels of unconjugated pteridines occurring in various biological fluids, such as urines, plasma and tissue culture media, a method has been developed for the separation and quantitative determination in the picomole range of ten 2-amino-4-hydroxy substituted pteridines. This method involves separation by high-pressure cation-exchange liquid chromatography and fluorescence detection of the eluted compounds at 450 nm. Optimal separation was obtained by isocratic elution with 3 mM phosphoric acid-7% methanol-1% acetonitrile at a flow-rate of 2 ml/min or with 1 mM ammonium dihydrogenphosphate pH 2.8-7% methanol-5% acetonitrile at a flow-rate of 1.5 ml/min. With either solvent, the order of elution of the compounds is: isoxanthopterin, pterin-6-carboxylic acid, xanthopterin, pterin-6-carboxaldehyde, D-erythro-neopterin, L-threo-neopterin, biopterin, 6-hydroxymethylpterin, pterin, 6-methylpterin. In addition, a systemic investigation of the effects of ammonium ion concentration and pH of the solvent as well as column temperature on the separation of these compounds was also conducted.


Fractionation of mannose-labeled neutral glycopeptides by QAE-Sephadex chromatography. Mannose-labeled cellular glycopeptides derived from human diploid fibroblasts (KL-2) were separated into two classes by QAE-Sephadex chromatography. High-voltage paper electrophoresis and Sephadex G-50 chromatography were used to characterize the glycopeptides further. At least five distinct neutral fractions were isolated with molecular weights ranging from 1050 to 2000 daltons. A linear gradient of ammonium acetate eluted a heterogeneous population of acidic glycopeptides. The use of QAE-Sephadex enables a single-step fractionation of both neutral and acidic glycopeptides on one column. The technique appears to be sensitive enough to distinguish growth-dependent alterations between growing and non-growing cells.


High-performance liquid chromatography of pirimiphos methyl and five metabolites. Pirimiphos methyl is an organophosphorus insecticide which is rapidly metabolised by plants and animals to several modified triesters and free hydroxyprimidines. A method is described for the determination by reversed-phase high-performance liquid chromatography of pirimiphos methyl and its five major metabolites in plasma and urine. Separations were performed by isocratic and gradient elutions from short columns packed with SAS-Hypersil, a relatively new column packing.


Differential effects of a partially purified preparation of slow-reacting substance of anaphylaxis on guinea pig tracheal spirals and parenchymal strips. The contractile effects of partially purified slow-reacting substance of anaphylaxis (SRS-A) and histamine were compared on isolated guinea pig tracheal spirals and parenchymal strips. Histamine was equally active on both isolated tissues in a concentration-related fashion. SRS-A (0.1--10.0 U/ml) produced a concentration-related effect on parenchymal strips, whereas the tracheal spiral was 100 times less sensitive to this mediator. The contractile activity of SRS-A on parenchymal strips was diminished by incubation with limpet arylsulfatase and antagonized by FPL 55712, a known SRS-A antagonist. SRS-A, further purified by high pressure liquid chromatography, also demonstrated this preferential activity on guinea pig parenchymal strips. These data are consistent with the hypothesis, based on previous in vivo observations, that SRS-A is a selective peripheral airway constrictor.


Synergistic interactions of physiologic increments of glucagon, epinephrine, and cortisol in the dog: a model for stress-induced hyperglycemia. To evaluate the role of anti-insulin hormone actions and interactions in the pathogenesis of stress-induced hyperglycemia, the counterregulatory hormones, glucagon, epinephrine, and cortisol were infused alone as well as in double and triple combinations into normal conscious dogs in doses that were designed to simulate changes observed in severe stress. Infusion of glucagon, epinephrine, or cortisol alone produced only mild or insignificant elevations in plasma glucose concentration. In contrast, the rise in plasma glucose produced by combined infusion of any two counterregulatory hormones was 50-215% greater (P < 0.005-0.001) than the sum of the respective individual infusions. Furthermore, when all three hormones were infused simultaneously, the increment in plasma glucose concentration (144+/-2 mg/dl) was two- to fourfold greater than the sum of the responses to the individual hormone infusions or the sum of any combination of double plus single hormone infusion (P < 0.001). Infusion of glucagon or epinephrine alone resulted in a transient rise in glucose production (as measured by [3-(3)H]glucose). While glucagon infusion was accompanied by a rise in glucose clearance, with epinephrine there was a sustained, 20% fall in glucose clearance. When epinephrine was infused together with glucagon, the rise in glucose production was additive, albeit transient. However, the inhibitory effect of epinephrine on glucose clearance predominated, thereby accounting for the exaggerated glycemic response to combined infusion of glucagon and epinephrine. Although infusion of cortisol alone had no effect on glucose production, the addition of cortisol markedly accentuated hyperglycemia produced by glucagon and(or) epinephrine primarily by sustaining the increases in glucose production produced by these hormones. The combined hormonal infusions had no effect on beta-hydroxybutyrate concentration. It is concluded that (a) physiologic increments in glucagon, epinephrine, and cortisol interact synergistically in the normal dog so as to rapidly produce marked fasting hyperglycemia; (b) in this interaction, epinephrine enhances glucagon-stimulated glucose output and interferes with glucose uptake while cortisol sustains elevations in glucose production produced by epinephrine and glucagon; and (c) these data indicate that changes in glucose metabolism in circumstances in which several counterregulatory hormones are elevated (e.g., "stress hyperglycemia") are a consequence of synergistic interactions among these hormones.


Different HLA-D associations in adult and juvenile rheumatoid arthritis. HLA-D typing was performed in 126 patients with juvenile rheumatoid arthritis. HLA-DW4, the antigen found in previous studies to characterize adult rheumatoid arthritis, had a significantly lower frequency in children with arthritis than in normal controls (P less than 0.04). By contrast, in children the antigens HLA-DW7 (P less than 0.03) and HLA-DW8 (P less than 0.01) were increased compared to controls. The antigen TMo, detected with homozygous typing cells from a child with juvenile rheumatoid arthritis, was found to be related to the cross-reactive specificities HLA-DW7 and DW11. 46% of the patients with persistent pauciarticular arthritis of childhood typed for the antigen TMo, compared to only 1% of normal controls. Thus the relative risk for persistent pauciarticular arthritis in relation to the presence of TMo was 67.7 (P less than 0.0001). These results provide evidence of fundamental differences between adult rheumatoid arthritis and arthritis of childhood. The latter group appears to include a population distinguishable clinically and characterized in these studies by the HLA-D determinant TMo.


Effects of prostaglandin cyclic endoperoxides on the lung circulation of unanesthetized sheep. Although prostaglandins E(2) and F(2alpha) have been suggested as mediators of the pulmonary hypertension seen after endotoxin infusion or during alveolar hypoxia, their precursors, the endoperoxides (prostaglandins G(2) and H(2)) are much more potent vasoconstrictors in vitro. In this study we compared the effects of prostaglandin (PG)H(2), a stable 9-methylene ether analogue of PGH(2) (PGH(2)-A), PGE(2), and PGF(2alpha) on pulmonary hemodynamics in awake sheep. The animals were prepared to allow for measurement of (a) lung lymph flow; (b) plasma and lymph protein concentration; (c) systemic and pulmonary vascular pressures; and (d) cardiac output. We also determined the effect of prolonged PGH(2)-A infusions on lung fluid balance and vascular permeability by indicator dilution methods, and by assessing the response of lung lymph. Both PGH(2) and PGH(2)-A caused a dose-related increase in pulmonary artery pressure: 0.25 mug/kg x min tripled pulmonary vascular resistance without substantially affecting systemic pressures. Both were 100 times more potent than PGE(2) or PGF(2alpha) in this preparation. PGH(2)-A, as our analysis of lung lymph and indicator dilution measurements show, does not increase the permeability of exchanging vessels in the lung to fluid and protein. It does, however, augment lung fluid transport by increasing hydrostatic pressure in the pulmonary circulation. We conclude: (a) that PGH(2) is likely to be an important mediator of pulmonary vasoconstriction; (b) its effects are probably not a result of its metabolites PGE(2) or PGF(2alpha).


C5 chemotactic fragment induces leukocyte production of tissue factor activity: a link between complement and coagulation. Complement-activated human plasma causes generation of tissue factor in human leukocytes. This phenomenon appears to be related to the fifth component of complement (C5) as demonstrated by the use of C5 deficient-plasma and suppression of activity with antibody to C5. Isolation of the chemotactic factor from activated serum or trypsinization of purified C5 reproduces the phenomenon. These data provide evidence for a direct link between complement products and activation of the coagulation system. Because chemotactic peptides from C5 can be generated by a variety of enzymes, our findings suggest a relationship between complement, coagulation, and inflammation.


Lymphocyte adherence in multiple sclerosis: effect of aspirin. Peripheral blood lymphocytes from multiple sclerosis (MS) patients form substantially greater numbers of rosettes with measles virus-infected human epithelial cells than do lymphocytes from healthy controls or from patients with other diseases. We have previously shown that prostaglandin E(1)-treated normal lymphocytes exhibit increased lymphocyte adherence, and thus behave like MS lymphocytes in this in vitro system. In this study we describe the effect of prostaglandin synthesis inhibition on lymphocyte adherence in both MS and control patients. Direct addition of aspirin or indomethacin to peripheral blood mononuclear cells from MS patients in vitro reduced lymphocyte adherence to control levels. Ingestion of therapeutic (anti-inflammatory) doses of aspirin (1 g, 4 times daily for 2 d) by MS patients resulted in reduction of lymphocyte adherence to levels seen in healthy controls. A single 325-mg dose of aspirin did not reduce lymphocyte adherence. A dose-dependent reduction in lymphocyte adherence was observed after single doses ranging from 650 mg to 1.3 g; duration of the effect was directly related to the aspirin dose. These observations indicate that treatment of MS patients with aspirin profoundly influences adherence of their lymphocytes to measles virus-infected cells and suggest that the altered cellular response, which results in increased lymphocyte adherence in MS patients, may be mediated by a prostaglandin-sensitive mechanism.


Hyperoxic attenuation of exercise-induced bronchospasm in asthmatics. To investigate the mechanism of exercise-induced bronchospasm, we measured specific airway conductance before and after exercise in 7 healthy normals, 12 asthmatics with intact carotid bodies, and 5 asthmatics who had had bilateral carotid body resection. The subjects breathed either air or oxygen (randomly assigned) during cycle ergometer exercise. Post-exercise bronchodilation was the usual pattern in normals, whereas post-exercise bronchospasm occurred in all asthmatics who breathed air during exercise. Oxygen breathing during exercise markedly attenuated the post-exercise bronchospasm in those asthmatics with intact carotid bodies, but had no significant effect in those without effect in those without carotid bodies. The attenuation of the bronchospasm with oxygen occurred with either incremental or constant load exercise of high intensity. The degree of attenuation did not correlate significantly with changes in end-tidal PCO2, maximum work rate, maximum exercise ventilation, or maximum heart rate. These studies indicate that oxygen attenuates exercise-induced bronchospasm in asthmatics through its action on the carotid bodies.


Cholic acid biosynthesis: the enzymatic defect in cerebrotendinous xanthomatosis. Cholic acid biosynthesis is defective in individuals with cerebrotendinous xanthomatosis (CTX) and is associated with the excretion of 5beta-cholestane-3alpha,7alpha, 12alpha,25-tetrol, an intermediate in the 25-hydroxylation pathway of cholic acid in CTX. To define the enzymatic defect in CTX, two suspected precursors of cholic acid, namely 5beta-[7beta-(3)H]cholestane-3alpha,7alpha, 12alpha-triol and 5beta-[24-(14)C]cholestane-3alpha,7alpha, 12alpha,24S,25-pentol were examined by both in vivo and in vitro experiments. A third precursor, 5beta-[7beta-(3)H]-cholestane-3alpha,7alpha, 12alpha,25-tetrol, was compared with them in vitro. In the in vivo experiments, each one of the labeled precursors was administered intravenously to two CTX and two control subjects. In the controls, 5beta-[7beta-(3)H]cholestane-3alpha,7alpha, 12alpha-triol as well as 5beta-[24-(14)C]-cholestane-3alpha,7alpha, 12alpha,24S,25-pentol were rapidly converted to labeled cholic acid. Maximum specific activity values were reached within 1 d after pulse labeling, followed by exponential decay of the cholic acid specific activity curves. In contrast, these two precursors differed widely when administered to two CTX patients. While 5beta-[24-(14)C]cholestane-3alpha,7alpha, 12alpha,24S,25-pentol was rapidly converted to [24-(14)C]cholic acid and yielded identical decay curves with those obtained in the control subjects, maximum specific activity values in [7beta-(3)H]cholic acid were much lower and peaked only on the second day after the injection of 5beta-[7beta-(3)H]cholestane-3alpha,7alpha, 12alpha-triol. Furthermore, an appreciable amount of (3)H label was present in the 5beta-cholestane-3alpha,7alpha, 12alpha,25-tetrol isolated from the bile of the subjects with CTX. In the in vitro experiments, three enzymes on the 25-hydroxylation pathway of cholic acid were examined in both control and CTX subjects. The rate of the 25-hydroxylation of 5beta-cholestane-3alpha,7alpha, 12alpha-triol in CTX patients was comparable to that in the controls. Similarly, the transformation of 5beta-cholestane-3alpha,7alpha, 12alpha,24S,25-pentol to cholic acid, catalyzed by soluble enzymes, proceeded at approximately equal rates in CTX and in control individuals. On the other hand, the rate of 5beta-cholestane-3alpha,7alpha, 12alpha,24S,25-pentol formation was about four times greater in the control subjects than in the CTX patients.The results of the in vivo as well as the in vitro experiments suggest that the site of the enzymatic defect in CTX is at the 24S-hydroxylation of 5beta-cholestane-3alpha,7alpha, 12alpha,25-tetrol. The relative deficiency of this hydroxylase in CTX patients, accompanied by the accumulation of its substrate in bile and feces, probably accounts for the subnormal production of bile acids in CTX patients.


Loop of Henle bicarbonate accumulation in vivo in the rat. We have carried out perfusion studies on hydropenic and bicarbonate-loaded rats to provide direct in vivo observations on bicarbonate accumulation in the short loops of Henle. Analysis of early distal tubular fluid was made during bicarbonate-free saline perfusion from the end proximal to the early distal site, documenting accumulation of "new" bicarbonate. During perfusion in hydropenic rats, steady-state bicarbonate concentrations were suggested by early distal values of approximately equal to mM, which were independent of perfusion rate and virtually indistinguishable from bicarbonate concentration measured during free flow when filtered bicarbonate was allowed to enter the loop. Thus, loop bicarconate accumulation was apparently sufficient to allow new bicarbonate to enter at a rate comparable to that delivered to the early distal site during free flow, recognizing of course that free-flow delivery rates are the result of complex components of filtration and bidirectional fluxes. In bicarbonate-loaded rats, however, bicarbonate accumulation rates although higher than in hydropenia, were much lower than free-flow delivery rates. Furthermore, early distal bicarbonate concentrations during bicarbonate loading fell as perfusion rate increased, presumably because of a limitation to increasing ionic bicarbonate entry.


The handling of immunoreactive vasopressin by the isolated perfused rat kidney. Using the isolated rat kidney perfused with an artificial medium containing glucose as the sole fuel, we studied the renal handling of immunoreactive arginine vasopressin (AVP) and determined the effect of various factors on the ability of the kidney to remove AVP. In control kidneys perfused with AVP at concentrations below 116 muU/ml, the organ clearance of AVP (OC(AVP)) was 1,145+/-47 (SE) mul/min, whereas glomerular filtration rate (GFR) averaged 515+/-37 mul/min. Filtration could thus account for up to 45% of the OC(AVP), the balance presumably being cleared from the peritubular circulation. Of the AVP filtered, only 38% could be recovered in the urine (urinary clearance AVP averaged 205+/-12 mul/min) suggesting that the balance was taken up by the tubular epithelium and degraded. Fractional excretion of filtered AVP rose significantly in the presence of anoxia and cold (10 degrees C) to 49 and 59%, respectively, but was not affected by ouabain or high levels of AVP (458+/-58 muU/ml). The OC(AVP) was not significantly altered by the absence of glucose in the perfusate, anoxia, or ureteral ligation, maneuvers that were associated with significant reductions in GFR. In these and the control experiments, there was a significant inverse correlation between GFR and peritubular clearance emphasizing the importance of the latter (r = -0.749; P < 0.001). Cold, ouabain, and high concentrations of AVP reduced the clearance of AVP by the kidneys. On the basis of these studies we conclude that the kidney clears AVP from the circulation via two pathways, glomerular clearance and peritubular clearance. This exposes both the luminal and contraluminal surfaces of the tubular cells to the hormone, allowing these cells to remove AVP from the filtrate and the peritubular compartment. Noteworthy is the observation that under several conditions when GFR falls reducing the glomerular clearance of AVP, peritubular clearance increases and the total clearance of AVP by the kidney remains constant.


Demonstration of a specific neutrophil receptor for a cell-derived chemotactic factor. The crystal-induced chemotactic factor, a cell-derived chemoattractant for neutrophils, binds specifically to a site on human neutrophils but not to erythrocytes or lymphocytes, suggesting a relationship between the presence of specific binding sites on the neutrophils and the ability to be chemotactically activated. The Scatchard analysis revealed an equilibrium dissociation constant at 37 degrees C of 0.446 micrometer and the presence of approximately equal to 6.44 x 10(5) binding sites for 125I-crystal-induced chemotactic factor per cell. Binding was not displaced by the synthetic chemotactic factors F-Met-Leu-Phe and Gly-His-Gly or by complement-activated plasma providing evidence of the specificity of the receptor.


Analysis of parathyroid hormone and its fragments in rat tissues: chemical identification and microscopical localization. After intravenous injection of [(125)I]-iodo-parathyroid hormone in the rat, uptake of the hormone was greatest in the liver and kidneys. Uptake was rapid, reaching a maximal concentration by 4 and 8 min, respectively. Extracts, prepared from both these organs at intervals soon after the injection of intact hormone, showed three main radioactive peaks when samples were subjected to gel filtration under protein-denaturing conditions. The first peak coeluted with intact hormone. The second eluted at a position corresponding to the carboxy-terminal fragments previously described in plasma, and the last eluted at the salt volume of the column. Microsequence analysis of the radioiodinated fragments, a method that has proved valuable for chemically defining the circulating fragments resulting from metabolism of injected hormone, showed that extracts of liver and kidney, prepared at 4 and 8 min after injection of the intact hormone, contained different fragments. The radioiodinated fragments in liver extracts were identical to those previously reported in the plasma of rats and dogs, fragments resulting principally from proteolysis between positions 33 and 34, and 36 and 37 of the intact hormone. Although the same fragments were also present in the kidneys, they constituted less than 15% of the amount present in the liver. More than 50% of the labeled renal fragments consisted of a peptide whose amino-terminal amino acid was position 39 of the intact hormone, a fragment not present in plasma. The rate of appearance of radioiodinated fragments that were chemically identical to those in plasma was more rapid in the liver than in plasma. Correlation of these chemical analyses with studies of the localization of (125)I by autoradiography showed that at the times when the intact hormone and the carboxy-terminal fragments comprised nearly all of the (125)I-labeled moieties in the tissues, the proximal convoluted tubules of the kidney and sinusoidal lining cells of the liver, which probably are Kupffer cells, contained the highest concentration of (125)I. Preferential localization of immunoreactive parathyroid hormone to these tissue sites also was shown by immunoperoxidase staining in studies with unlabeled hormone. Our results suggest that, unless multiple renal mechanisms are present for release of hormonal fragments, one of which releases the circulating fragments preferentially, the liver, rather than the kidney, is principally responsible for generating the carboxy-terminal fragments in plasma after injection of intact hormone, and the Kupffer cells may contain the enzymes that hydrolyze parathyroid hormone.


Electrophysiologic properties of coffee in man. There is no information on the effects of coffee on the human conduction system. His bundle electrograms were obtained in 12 patients before and 20 minutes after the ingestion of coffee containing 150 mg caffeine. Antegrade and retrograde refractory periods were obtained with the extrastimulus method. The effective and functional refractory period of the atrioventricular node decreased after coffee ingestion. This improvement in conduction is probably mediated by a release of catecholamines.


Variability of laminar patterns in the human lateral geniculate nucleus. The structure of the human lateral geniculate nucleus has been studied on serial Nissl stained sections from 57 human brains. Most of the brains were from neurologically normal individuals and were obtained during routine autopsy procedures. The laminar arrangement within the human nucleus is surprisingly variable. It is always possible to recognize a small segment with two layers (the monocular segment), one with four layers and one with six layers. Often an 8-layered segment can also be seen. The posterior half of the nucleus, within which central vision is represented, is made up mainly of six layers, and is the least variable part of the nucleus. Here the layers lie roughly parallel to each other. In the anterior half of the nucleus the laminar arrangement is more variable, and the layers often form complex and irregular interdigitations with each other. The 8-layered segment varies greatly in size and may be absent. It generally lies at the edge of the 4-layered segment not, as might have been expected, within the borders of the 6-layered segment. In many parts of the nucleus nerve cells are organized in short rows that run perpendicular to the layers; also, individual nerve cells are elongated in this direction. This neuronal orientation follows the lines of projection that are defined by the borders of a zone of retrograde degeneration, and also corresponds to the orientation of a cellular discontinuity that probably is the geniculate representation of the blind spot. Thus, we conclude that the neuronal orientation indicates the lines of projection within the nucleus.


A Golgi study of the early postnatal development of the visual cortex of the hooded rat. Although neuroanatomical plasticity has been demonstrated in the rat visual cortex, no systematic data on the dendritic development of the area are available. In the present study, the visual cortex of hooded rats at 1, 3, 5, 7, 10 and 15 postnatal days of age (P1-P15) was impregnated with the rapid Golgi method. The cortex was divided into the superficial layers, II-IV, and the middle layer V. At P1, pyramidal neurons had apical shafts and the beginning of the apical terminal arch. Analysis of both basilar and oblique dendritic number showed that pyramidal neurons of the middle layer developed more quickly than those in the superficial layers. The number of lower order basilar dendritic branches reached asymptote over the examined time period, whereas the higher order branches were still increasing in number but at a decelerating rate by P15. Dendrites at all ages exhibited varicosities which were especially prominent on the thin dendritic branches of the earlier ages. Some thin, filamentous processes, termed protospines, were found on dendrites and cell bodies at P1 to P5. They seemed to decrease by P7, when a few mature spines appeared. Spines increased in number on days P10 and P15. A comparison of the data from this study with quantified Golgi studies in adult rats indicates that by P10 and P15 the number of basilar branches is in the range seen in the adult.


An electron microscope study of the early postnatal development of the visual cortex of the hooded rat. Synaptic plasticity in response to environmental events has been clearly demonstrated in the visual cortex of the rat, but no detailed data concerning the course of early synaptogenesis in this area are available. In this study, synaptogenesis in the visual cortex of hooded rats at 1, 3, 5, 7 and 10 postnatal days of age (P1-P10) was examined with electron microscopy. The cortex was divided into the molecular layer, the superficial layers (II-IV) and deep layers (V-VI). In the visual cortex at P1, very few synapses are present in the molecular and deep layers and virtually none in the yet undifferentiated layers II-IV that compose the cortical plate at this age. The synapses that are present are axodendritic and often symmetrical with little membrane thickening and few vesicles. Axosomatic synapses were seen as early as P3 but very rarely. There are marked increases in axodendritic synaptic density and maturity with increasing age. By P7 and P10, many synapses appear mature in form and the majority can be classified a symmetrical. Axospinal synapses first appeared at P7 and were more frequent by P10. However, this classification was somewhat uncertain since no spinal apparatus was detected. Rate of synaptogenesis appeared to increase over the ages studied and showed no signs of leveling off except in the deep layers. Synaptic length was extremely variable and did not change systematically with age.


Properties of superior colliculus neurons in the golden hamster. A laminar representation of sensory modalities was present in the hamster superior colliculus (SC) with upper laminar cells being exclusively visual, while intermediate and deeper layer cells were visual, somatic, acoustic or multimodal. Receptive field (RF) properties of visual SC cells were studied quantitatively with stationary spots and moving bars of light of various sizes, velocities and directions of movement. The most effective stimuli were usually less than half of the diameter of the RF. Increasing stimulus size beyond a critical range produced progressively lower discharge rates even though the stimulus was confined within the borders of the RF. Low velocities (10-50 degrees/sec) were most frequently optimal for both upper and lower laminar cells and the majority of cells were directionally selective. Movements in the upper nasal direction usually elicited the highest discharge rates and were thus preferred most frequently. However, directional preferences often could be minimized, or obliterated, by employing nonoptimal stimulus sizes and/or velocities. Most intermediate and deeper laminar somatic cells could be activated by gentle cutaneous stimuli and a general somatotopic plan, which was in register with the overlying visuotopy, was noted. Cells optimally, or solely, activated by noxious stimuli were also located, but RFs of such cells were extensive and a somatotopic plan was not apparent. Although species differences are apparent, the similarities between the organization and the RF properties of SC cells of the hamster and distantly related species are striking. Apparently the same SC system is adaptive in diverse species despite the very different behavioral repertories of these animals and their different ecological niches.


The formation of afferent patterns in the somatosensory cortex of the neonatal rat. In the rat, the clustered pattern of thalamocortical afferent terminals to the "barrel field" portion of primary somatosensory cortex replicates the arrangement of vibrissae on the face, and the pattern of the terminals can be altered by the removal of vibrissae on the face, and the pattern of the terminals can be altered by the removal of vibrissae at birth (Killackey et al., '76). These patterns of terminals were studied using enzyme succinic dehydrogenase (SDH) because in somatosensory cortex the activity levels of SDH closely correspond to the patterns of thalamocortical afferent terminals. The present experiments show that the pattern of high SDH segmentation in the portion of layer IV of somatosensory cortex that is related to the vibrissae develops during postnatal Day 3 through 6. Activity related to the centers of individual clusters is first visible, with indistinct boundaries. At later times the edges of individual clusters become apparent. Further, in animals with Row C of vibrissae removed at birth, the abnormal SDH segmentation in somatosensory cortex develops with a time course similar to that of normal animals. At ages when edge boundaries are first distinct, a fused band corresponding to the removed row of vibrissae is present. Thus the aberrant organization seen in the adult cortex is the result of an abnormal initial development, not a later reorganization from a normal pattern. And indeed, vibrissae removal at Day 5 or 6 does not result in an aberrant cortical SDH pattern. Finally, after removal of all five rows of mystacial vibrissae at birth, the cortical SDH pattern seen at postnatal Days 6 and 7 consists of five bands in place of the normally present five rows of clusters. This may indicate that closer relationships exist between vibrissae within one row than vibrissae in adjacent rows.


Vibrissae representation in subcortical trigeminal centers of the neonatal rat. In the neonatal rat differential activity levels of the metabolic enzyme succinic dehydrogenase (SDH) reveal intricately detailed sgementation in the neuropil of the spinal and principal trigeminal nuclei of the brainstem and in the ventrobasal complex of the thalamus. The segmentation occurs in the portions of these nuclei that electrophysiological evidence has indicated to be related to the mystacial vibrissae and sinus hairs on the face of the rat. Indeed, the pattern of segmentation in each nucleus replicates the topographic distribution of the vibrissae and sinus hairs. Further, within the spinal trigeminal nucleus, there appear to be two distinct representations of the vibrissae, one in the subnucleus caudalis and a second in the subnucleus interpolaris. Examination of these patterns of segmentation indicates that the large mystacial vibriaase and sinus hairs on the face of the young rat are somatotopically represented three times within the trigeminal complex, as straight cylinders of neuropil, and once in the ventrobasal complex, as curved cylinders of neuropil. Neonatal vibrissae damage leads to an aberrant organization of the segmentation in the spinal trigeminal nucleus and the ventrobasal complex. In the spinal trigeminal nucleus, the SDH activity in areas associated with damaged vibrissae is of a lower than normal density, and patterns are indistinct. However, rows of clusters associated with the adjacent normal vibrissae are apparent and appear to be enlarged. In the ventrobasal complex, vibrissae damage results in bands of normal density SDH activity where rows of segmented clusters would normally be present. Comparison of these data to the cortical data in the previous paper (Killackey and Belford, '79) indicates that cortical and nuclear structures can have aspects of their development controlled by similar mechanisms.


The posterior lateral line lobe of certain gymnotoid fish: quantitative light microscopy. The posterior lateral line lobe of the wave species of gymnotoid fish was investigated with the Golgi technique. The posterior lobe has a laminar structure and contains II cell types differentially distributed in the varous laminae (fig. 13). The major laminae, from ventral to dorsal are the deep fiber layer, containing multipolar neurons; the deep neuropil layer, containing ovoid neurons and a sub-lamina of spherical cells; the granule cell lamina, containing two types of granule cell; the plexiform laminae; the polymorphic cell lamina, containing basilar pyramids, non-basilar pyramids, giant fusiform cells, and polymorphic cells; the stratum fibrosum; the molecular lamina, containing neurons of the ventral molecule layer and stellate cells. The spherical cells are regularly distributed in their sub-lamina and appear to receive one type of primary afferent input. Another type of primary afferent input ends in the deep neuropil and granule layers, in proximity to the basilar dendrites of the granule cells and the basilar pyramids. The basilar pyramidal cell spatially alternates with the non-basilar pyramidal cell, so that the basilar dendritic trees of nearest-neighbour basilar pyramids show almost no overlap. Descending input to the posterior lobe ends in the molecular layer, in proximity to apical dendrites of both pyramidal cells, giant fusiform cells, polymorphic cells, and one type of granule cell. There are three afferent fiber systems in the molecular layer, one running transversely, one longitudinally, and one vertically. Local circuity in the posterior lobe is precisely organized and involves projections of granule cells onto overlying pyramidal cells. The polymorphic cell may also be involved in the intrinsic circuits of the posterior lobe.


An ultrastructural study of Schwann cell response to axonal degeneration. Adult Sprague-Dawley rats were used to prepare isolated and distal segments of ovarian, vagus and saphenous nerves. Isolated segments were prepared by cutting between double ligatures followed by retracting and anchoring the proximal and distal segments 0.5 cm or more away from the isolated piece. After various intervals between 3 and 123 days and rats were perfused with buffered glutaraldehyde and nerves were processed for electron microscopic examination. Cross sections of isolated and distal segments of all nerves displayed an abundance of mostly circular profiles containing microtubules and filaments that were indistinguishable from those in unmyelinated nerve fibers. Many such profiles were nested against perinuclear Schwann cell cytoplasm, others were present in isolated clusters enclosed only by basement membrane. Examples of continuity between the nerve-like profiles and perinuclear cytoplasm of Schwann cells were found in longitudinal sections. In time, the number of such processes gradually diminished, but 123 days after transection they still could be found. It was concluded that Schwann cells in isolated and distal segments of transected nerves develop great numbers of small cylindrical processes that are arranged in bundles parallel to the long axis of the degenerated nerve. These processes had an arrangement of cytoplasmic organelles similar to axons of the proximal segment and the contralateral nerve. However the processes differed from most mature axons in that they were not ensheathed by Schwann cell cytoplasm with a mesaxon.


A regional specialization in the opossum's retina: quantitative analysis of the ganglion cell layer. The distribution of ganglion cells in the opossum's retina was determined from flat-mounted preparations stained with cresyl-violet. The retinal area is 109 mm2 (SD = 16 mm2). Maps of ganglion cell density were made from retinae of seven animals. In all maps iso-density lines were approximately concentric, showing a slight elongation towards the nasal region. Cell density varied from 400 cells/mm2 at the extreme periphery to 2,900 cells/mm2 in the region of highest count, the are centralis. The center of this region lies 1.85 mm (26.3 degree) temporal to the center of the optic nerve head. The average total number of ganglion cells is 77,384 (SD = 10,173). Based upon soma diameter histograms ganglion cells were classified into three groups, showing at area centralis peaks at 7 micrometer, 12 micrometer and 15 micrometer respectively. Cell soma diameter ranged from 6 micrometer to 21 micrometer, larger values being observed at the periphery.


The organization of the electromotor nucleus and extraocular motor nuclei in the stargazer (Astroscopus y-graecum). The organization of the oculomotor and electromotor systems was examined in the stargazer, a teleost. The electromotor system in these animals is a derivative of the oculomotor system. The extraocular motor nuclei and nerves consist of approximately equal numbers of motoneurons and axons (about 100 per muscle). In contrast, electromotor axons appear to branch several times within the intracranial portion of the IIIrd nerve. The topographical organization of the motoneurons was examined using retrograde transport of horseradish peroxidase injected into the electric organ or eye muscles. Electromotor and oculomotor neurons form distinct populations. Each electric organ receives a strong ipsilateral and a weak contralateral innervation. Individual eye muscles receive unilateral innervations with the expected laterality. Within the oculomotor nucleus there is some topographical separation of motoneurons innervating each muscle. Antidromic field potentials confirm the identity of the electromotor nucleus.


Evidence for a critical period of neuronal trophism late in the development of the chick visual system. Chick embryos and young hatchling chicks have undergone unilateral retinal ablation and the brains were subsequently prepared for modified Fink-Heimer staining to detect degenerating axoplasm. Additional animals have been injected with tritiated proline or combinations of tritiated proline and tritiated fucose. In examining the patterns of degeneration and labelling produced, it appears that during the stage 40-44 period of embryonic life there is a critical period during which damage to retinal axons produces degeneration not only in the direct targets of the optic tract (primary centers), but also in a number of nuclei not projected to by the optic tract (secondary centers). By examining various survival times following retinal ablation and animals of varying embryonic and post-hatch age, we have concluded that retinal ablation during the critical period causes a rapid and fulminant degeneration in the secondary centers because they are all targets of the projection from the optic tectum, itself a prime target of the optic projection. Presumably, interruption of the retino-tectal projection during the critical period disrupts some as yet undefined trophic relationship between these two sets of neurons. Our studies show that the observed degenerative reaction in the secondary centers occurs most strongly in chick embryos between stage 40 and stage 44. This period coincides with the onset of synaptogenesis in the retino-tectal system (Rager, '76a,b). We therefore hypothesize that during this period some property or influence is passing from the retinal to the tectal neurons such that interruption of this process leads to death of the tectal neurons.


Electron microscopic studies on the lamprey meninges. Lamprey meninges were revealed to consist of four layers. The I (outermost) layer was made up of densely packed collagen fibrils and microfibrils with fibrocytes interposed between them. It contained a vascular network (epidural vascular plexus). The II layer was a cellular layer, consisting of two parts: the outer, membranous and the inner, spongy part. The membranous part was composed of two sheets of type 1 cells which were held together by desmosomes, gap junctions and tight junctions. Continuous basal laminae were found on both the outer and the inner surface of the membranous part. The spongy part consisted of type 2 cells, collagen fibrils and microfibrils, which were loosely arranged. Type 2 cells were joined together with each other, or with type 1 cells, by gap junctions. These cells lacked a basal lamina covering. The III layer was a peculiar thick layer, being mostly occupied by fine filamentous matrix substance with scattered round cells, fibroblasts and microfibrils. The IV (innermost) layer was a thick fibrous one, consisting of two parts: the outer, vascular and the inner, loose part. The former consisted of a vascular network (perineural vascular plexus) and thick bundles of microfibrils which frequently contained a dense filamentous core. The latter was composed of loosely arranged collagen fibrils. The inner part of the I layer (inside the epidural vascular plexus) was considered to correspond to the true dura, and the II and IV layer to the arachnoid and pia in the meninges of higher animals, respectively. Characteristically the subarachnoid space was absent in lamprey meninges, and instead a peuliar "gelatinous" layer, III layer, was present between the II and IV layer.


The generation of neurons involved in an early reflex pathway of embryonic mouse spinal cord. The generation of lateral motor neurons (LMNs), interneurons and dorsal root ganglion (DRG) neurons of the cervical mouse spinal cord has been investigated by [3H]thymidine autoradiographic techniques. This investigation has two main objectives: (a) to determine on which embryonic days these three neuronal populations are born, and (b) to investigate the possibility that the neurons comprising early reflex circuits might be formed by a retrograde temporal sequencing of generation. LMNs are the first neurons generated in the cervical spinal cord. They arise between E8.8 and E11.5, and approximately 90% of these cells are born within a 36-hour period between E9 and E10.5. The earliest time of origin for interneurons is on E9.5, and those cells which are generated between E9.5 and E10.5 cluster in two distinct regions of the adult spinal cord. One of these regions is the lateral portions of laminae IV through VI; this appears to be the location of many ipsilateral association neurons. DRG neurons begin to arise on E9.5 and their generation is completed by E14. There is a trend within the DRG population for large neurons to be born before small neurons. Those cells with diameters of 40 micron or greater reach their generation peak on E10.5, while those smaller than 40 micron arise in the greatest numbers on E12. The findings of other investigations have provided evidence for a retrograde sequence of synaptic closure in the formation of the early disynaptic forelimb reflex pathway. The temporal difference in synapse formation in the terminal fields of DRG and association neurons is discussed in terms of our observation that both of these populations appear to have similar generation times. We suggest that factors responsible for the delayed synaptic closure of DRG afferents include the greater distances and the degree of collateralization which these afferents must undergo in order to establish their terminal fields. Finally, we discuss the possibility that the temporal sequence of neuronal generation and factors involved with the growth of neurites combine to produce a retrograde sequence of synaptic closure in the early disynaptic forelimb reflex pathway of mouse spinal cord.


Anomalous ipsilateral retinotectal projections in Syrian hamsters with early lesions: topography and functional capacity. Retinotectal topography, response properties of neurons in superior colliculus, and visual orienting behavior were studied in hamsters whose superior colliculi were innervated by one or the other of two types of anomalous ipsilateral projections. For the first type, an abnormally large uncrossed projection was created by monocular enucleation on the day of birth. This projection extended over the superficial part of the rostral half of the colliculus. The upper visual field was represented medially, and the lower visual field laterally, which corresponds to a normal projection. The rostrocaudal axis was disordered, but showed a slight tendency for nasal visual field to be represented rostrally and temporal field caudally; this tendency corresponds to an inversion of the normal ipsilateral projection, fitting instead the pattern of a contralateral projection. For the second type of anomalous ipsilateral projection, an abnormal intertectal decussation of optic tract fibers was created by neonatal ablation of the superficial layers of one superior colliculus and removal of the ipsilateral eye (Schneider, '73). Retinotectal topography observed in this recrossing projection was predominantly mirror-symmetric to the normal contralateral projection; however, some distortions in retinotopic order were observed, including misplaced fields and local inversions of the mirror-symmetric topography, and distortions of local magnification factor. Response properties of single units found medially in the left colliculus were similar to those found in normal colliculus. Units found more laterally were underresponsive, showing response decrements with repeated stimulation which is abnormal for units in the superficial gray, and many had abnormally large receptive fields. This physiological pattern was reflected in the pattern of errors made in visual orienting to small targets. It was concluded that polarity cues exist in the tectum sufficient to order the terminals of the retinotectal projection independent of the direction of fiber arrival or order in the optic tract as it enters the tectum. In addition, the functional competence of the abnormal recrossing retinotectal projection has been demonstrated by both electrophysiological and behavioral methods.


Ultrastructural development of Rohon-Beard neurons: loss of intramitochondrial granules parallels loss of calcium action potentials. We have examined the ultrastructure of the cell body of a vertebrate spinal neuron, the Rohon-Beard cell of Xenopus laevis, at four stages during its development (Nieuwkoop and Faber stages: 22, 29/30, 37/38 and 42). At this time it has attained its electrical excitability and the action potential mechanism in the cell body is maturing through a sequence of stages in which the inward current is carried by Ca++ (stages 20-25), later by Ca++ and Na+ (stages 25-40), and finally by Na+ (stages 40-51) (Spitzer and Baccaglini, '76; Baccaglini and Spitzer, '77). There is a change in the abundance and distribution of the organelles in the perikaryon during this period, characteristic of other developing neurons. Mitochondria and Golgi apparatus become localized progressively more in the interior of the cells, and rough endoplasmic reticulum progressively more to the periphery where it often appears in orderly tiers parallel to the plasma membrane. The mitochondria contain dense intramitochondrial granules which are known in other cells to contain concentrations of divalent cations. The number of granules declines over the course of the developmental period studied. The presence of the intramitochondrial granules was examined quantitatively because electrophysiological data indicate that the amount of Ca++ entering the cells in early stages should raise the internal Ca++ concentration by several orders of magnitude, and that Ca++ is rapidly sequestered (Baccaglini and Spitzer, '77). A minimum of 200 mitochondrial profiles from at least four Rohon-Beard cells were scored for the presence of dense intramitochondrial granules at each stage studied. In stage 22 Rohon-Beard cells 75 +/- 5% (mean +/- SD, n = 4) of the mitochondrial profiles scored contained granules; in stage 29/30, 56 +/- 10% (n = 7); in stage 37/38, 3 +/- 3% (n = 5); and in stage 42, 0.5 +/- 0.25% (n = 4). Therefore, dense intramitochondrial granules, an indication of calcium accumulation in mitochondria, decrease in parallel with the loss of the Ca++ component of the inward current of the action potential in Rohon-Beard neurons.


Efferent projections from temperature sensitive recording loci within the marginal zone of the nucleus caudalis of the spinal trigeminal complex in the cat. The efferent projections from nucleus caudalis of the spinal trigeminal complex in cats were studied with retrograde and anterograde axonal transport techniques combined with localization of recording sites in the thalamus and marginal zone of nucleus caudalis to innocuous skin cooling. Results showed brainstem projections from nucleus caudalis to rostral levels of the spinal trigeminal complex, to the ventral division of the principal trigeminal nucleus, the parabrachial nucleus, cranial motor nuclei 7 and 12, solitary complex, contralateral dorsal inferior olivary nucleus, portions of the lateral reticular formation, upper cervical spinal dorsal horn and, lateral cervical nucleus. Projections to the thalamus included; a dorsomedial region of VPM (bilaterally) and to the main part of VPM and PO contralaterally. Neuronal activity was recorded in the dorsomedial region of VPM to cooling the ipsilateral tongue. HRP injections in this thalamic region retrogradely labeled marginal neurons in nucleus caudalis. These results show that marginal neurons of nucleus caudalis provide a trigeminal equivalent of spinothalamic projections to the ventroposterior nucleus in cats.