Corpus allatum regulation during the metamorphosis of Periplaneta americana: Axon pathways

The anatomy of the retrocerebral complex was studied after supravital staining with methylene blue, and axonal tracts within the corpora allata (CA) were traced after applying the CoCl₂ technique together with Timm's sulfide-silver enhancement. Cobalt chloride fills of the nerves to and from the CA revealed two major sources of innervation: the brain and the subesophageal ganglion. Three cell clusters in the brain contribute axons that reach each nervus corporis allati I (NCA I) and, apparently, pass to or beyond the CA. These are: a cluster of 8 to 12 cells in the contralateral pars lateralis, a cluster of 16 to 20 cells in the ipsilateral pars lateralis, and a cluster of 50 to 60 cells in the contralateral pars intercerebralis. PAF-stained sections of other brains revealed a corresponding number of PAF-positive cells in these same regions. The medial and lateral neurons arborize in the neuropile adjacent to the pars intercerebralis, and may associate there. The lateral group also arborizes extensively in the neuropile surrounding the pedunculus of the mushroom body. At least four cell bodies located antero-ventrad in the subesophageal ganglion send axons to the CA via each nervus corporis allati II (NCA II).To determine possible inhibitory pathways to the CA, the NCA I, NCA II, and postallatal nerves of last instar larvae were severed; either singly, or in combination. Additional experiments were performed on last instar larvae to substantiate that superlarvae were a direct result of an enhanced or sustained juvenile hormone titre. These experiments included: implanting two or more CA, extirpating one CA, or applying 100 μg of Altosid topically onto allatectomized larvae. The experiments indicated that only NCA I is an inhibitory pathway and that superlarvae were a direct consequence of CA activation. NCA II does not seem to provide the CA with an essential excitatory innervation; when it and NCA I are severed a supernumerary apolysis will still result. Some of the cells in the brain stainable by the CoCl₂ method are most probably identical to those that are PAF-positive. These cells may inhibit the CA in last instar larvae via neurosecretomotor junctions.     

Correlation between DNA synthesis and intracellular NAD in cultured human leukemic lymphocytes.

The NAD⁺ level in lymphocytes obtained from an individual with acute monocytic leukemia increased five-fold and then remained constant when the cells were adapted to growth in suspension culture. When the NAD⁺ level of established cells was lowered by means of a nicotinamide-poor medium or by the action of 1-methyl-1-nitrosourea, there was a concomitant decrease in the rate of DNA synthesis. These results indicate that there is a direct correlation between intracellular NAD⁺ and the synthesis of DNA in cultured leukemic lymphocytes. However, the exact nature of the relationship remains speculative.     

Studies on the regulation of leucine catabolism: Mechanism responsible for oxidizable substrate inhibition and dichloroacetate stimulation of leucine oxidation by the heart

In the absence of any other oxidizable substrate, the perfused rat heart oxidizes [1-¹⁴C]leucine to ¹⁴CO₂ at a rapid rate and releases only small amounts of α-[1-¹⁴C]ketoisocaproate into the perfusion medium. The branched-chain α-keto acid dehydrogenase complex, assayed in extracts of mitochondria prepared from such perfused hearts, is very active. Under such perfusion conditions, dichloroacetate has almost no effect on [1-¹⁴C]leucine oxidation, α-[1-¹⁴C]ketoisocaproate release, or branched-chain α-keto acid dehydrogenase activity. Perfusion of the heart with some other oxidizable substrate, e.g., glucose, pyruvate, ketone bodies, or palmitate, results in an inhibition of [1-¹⁴C]leucine oxidation to ¹⁴CO₂ and the release of large amounts of α-[1-¹⁴C]ketoisocaproate into the perfusion medium. The branched-chain α-keto acid dehydrogenase complex, assayed in extracts of mitochondria prepared from such hearts, is almost completely inactivated. The enzyme can be reactivated, however, by incubating the mitochondria at 30 °C without an oxidizable substrate. With hearts perfused with glucose or ketone bodies, dichloroacetate greatly increases [1-¹⁴C]leucine oxidation, decreases α-[1-¹⁴C]ketoisocaproate release into the perfusion medium, and activates the branched-chain α-keto acid dehydrogenase complex. Pyruvate may block dichloroacetate uptake because dichloroacetate neither stimulates [1-¹⁴C]leucine oxidation nor activates the branched-chain α-keto acid dehydrogenase complex of pyruvate-perfused hearts. It is suggested that leucine oxidation by heart is regulated by the activity of the branched-chain α-keto acid dehydrogenase complex which is subject to interconversion between active and inactive forms. Oxidizable substrates establish conditions which inactivate the enzyme. Dichloroacetate, known to activate the pyruvate dehydrogenase complex by inhibition of pyruvate dehydrogenase kinase, causes activation of the branched-chain α-keto acid dehydrogenase complex, suggesting the existence of a kinase for this complex.     

The regulation of ornithine aminotransferase synthesis by glucagon in the rat.

Ornithine aminotransferase (OAT) from rat liver mitochondria was purified to homogeneity. A monospecific antiserum against the enzyme protein was prepared in rabbits. Immunotitrations were performed on OAT present in crude mitochondrial extracts obtained from the livers and kidneys of rats in several hormonal and dietary states. No evidence was found for the existence of an immunologically reactive but enzymatically inactive form of OAT. The relative rate of enzyme synthesis in vivo was studied by pulselabeling rats with [4, 5-³H]leucine, isolating the enzyme protein by immunoprecipitation, and dissociating the immunoprecipitates on sodium dodecyl sulfate-acrylamide gels. Nine hours after a single subcutaneous injection of a glucagon oil emulsion, a 3-fold increase in OAT activity and a 12-fold increase in the synthetic rate of the enzyme were observed. Serine dehydratase activity increased on a time-course very similar to that of OAT following glucagon injection. These increases occurred only on low (0–12.5%) protein diets. At higher levels of dietary protein (30% and up), no further stimulation of OAT synthesis by glucagon was observed. Administration of actinomycin D within the first 2 h after glucagon injection resulted in an inhibition of OAT induction. When the administration of the antibiotic was delayed until 4 h after glucagon, no inhibition of OAT induction was observed. Glucose repression of the glucagon induction of the enzyme in hepatic mitochondria was demonstrated to be the result of a rapid inhibition of OAT synthesis.     

Mechanism responsible for the hypoglycemic actions of dichloroacetate and 2-chloropropionate

Dichloroacetate, an activator of the pyruvate dehydrogenase complex, is known to lower blood glucose, lactate, pyruvate, and alanine when given to diabetic and 24 h fasted rats. Under certain conditions, especially when pyruvate carboxylase is made rate limiting for want of bicarbonate, dichloroacetate effectively inhibits glucose synthesis from lactate by isolated hepatocytes. 2-Chloropropionate also activates the pyruvate dehydrogenase complex, lowers blood glucose, lactate, and pyruvate in 24 h fasted rats, but stimulates gluconeogenesis from lactate or alanine by isolated hepatocytes. Dichloroacetate is catabolized to glyoxylate and thence to oxalate by liver cells, whereas 2-chloropropionate cannot be catabolized to these products. Glyoxylate and oxalate are potent inhibitors of glucose synthesis from lactate, pyruvate, and alanine, but not from dihydroxyacetone. Inhibition is much more pronounced in a bicarbonate-deficient medium, in which pyruvate carboxylase is probably rate limiting for gluconeogenesis. It seems likely, therefore, that the inhibition of lactate gluconeogenesis by dichloroacetate is actually caused by oxalate, which inhibits pyruvate carboxylation. Nevertheless, the major effect of dichloroacetate, and probably the sole effect of 2-chloropropionate, on blood glucose concentration is to limit substrate availability in the blood for hepatic gluconeogenesis. Since oxalic acid stone formation and renal dysfunction may prove to be side effects of any therapeutic application of dichloroacetate, we suggest that further studies on the treatment of hyperglycemia and lactic acidosis with pyruvate dehydrogenase activators be carried out with 2-chloropropionate rather than dichloroacetate.     

Programmed autophagocytosis accompanying conjugation in the ciliate Stylonychia mytilus

Conjugation and postconjugant development in Stylonychia is accompanied by a period of approximately 80 hr during which the cells are unable to ingest food. This period is one of considerable synthetic activity, encompassing important portions of the development of the new macronucleus. Light- and electron-microscopic observations of conjugating pairs and exconjugant cells reveal a process of autophagocytosis that may provide the supplies of energy (and precursors) necessary for postconjugant developmental events. Small autophagosomes (AP) form in conjugants; degenerating mitochondria are prominent among the inclusions observed in these bodies. Soon after separation of pairs, large dense AP appear, apparently by coalescence and condensation of the smaller AP. These “mature” AP give only a slight acid phosphatase reaction. The number of AP slowly declines during postconjugant development; about 60 hr after separation all the AP have been digested. Several observations suggest that this autophagocytosis is part of the developmental program initiated soon after mating begins: (1) The first indications of AP formation occur while conjugating pairs are still able to feed, and thus cannot be attributed to the stress of starvation; (2) formation of large numbers of AP is rather abrupt, whereas their dissolution is very gradual, covering most of the nonfeeding period; and (3) the pattern of AP formation and dissolution is similar in cells whose new macronuclear development has been prevented by brief hydroxyurea treatment.     

Ca2+-stimulated ATPase during the early development of parthenogenetically activated eggs of the sea urchin Paracentrotus lividus

A Ca²⁺-stimulated ATPase shows fluctuations in the activity in parthenogenetically activated sea urchin eggs very similar to those described earlier for fertilized eggs. Besides activity peaks in the first part of a cell cycle the enzyme activity increases when the mitotic apparatus (MA) or MA-like structures like monasters or cytasters are formed. A possible function of the enzyme in the assembly of the MA is discussed.     

Melanogenesis in cultures of peripheral nervous tissue. II. Environmental factors determining the fate of pigment-forming cells

Spinal ganglia from 4- to 7-day [Stage 23–30; Hamburger and Hamilton (1951) J. Morphol.88, 49–92] chicken embryos were cultured in vitro to investigate the effect of various environmental conditions on cell differentiation. Culture morphology (i.e., degree of dispersion of the explanted ganglia, survival of neurons, and outgrowth of axons) was observed to depend upon several factors including: (1) the age of the explanted ganglia, (2) the presence or absence of nerve growth factor (NGF), and (3) the nature of the substratum on which the cultured tissue resides. These observations enabled us to disturb the association of neurons with the other cells in ganglion cultures and thereby modulate the differentiation of adventitious melanocytes. Thus, in medium permissive for melanogenesis, melanocytes appear when the association between neurons and small stellate nonneuronal cells in the ganglion is disrupted. This disruption is most extensive (1) when young (Stage 26–27, 5-day) ganglia are explanted on plastic substrata, in the initial absence of NGF, and (2) when cells from enzyme-dissociated ganglia are cultured on plastic substrata. In comparable media, pigment cell differentiation is not observed when the association between neurons and small stellate cells is preserved. Such associations tend to endure (1) in developmentally older (Stage 30+, 7- to 8-day) ganglia or (2) when ganglia are cultured on agar or fibroblast substrata. We conclude that loss of association between neurons and the nonneuronal cells in young ganglia is necessary for the latter to undergo melanogenesis in vitro.     

Alteration of liver plasma membrane protein conformation by bovine growth hormone in vitro

The response of liver plasma membranes prepared from hypophysectomized, bovine growth hormone-treated hypophysectomized, and normal rats to addition of bovine growth hormone in vitro were studied by circular dichroism and spectrofluorometry. Membranes of hypophysectomized but not normal or treated rats showed increased negative ellipticity in the presence of bovine growth hormone (10^?9) without any change in trough position; at higher concentrations (10^?7?10^?6, m) there was less negative ellipticity. Membranes of hypophysectomized, normal, and growth hormone-treated rats all showed decreased emission of fluorescence and a small shift of the emission peak from 333 to 338 nm in the presence of bovine growth hormone (10^?17?10^?7, m). The excitation of fluorescence was quenched by bovine growth hormone. Polarization of excitation of fluorescence was unchanged.     

Calcium ion-flux across phosphatidylcholine membranes mediated by ionophore A23187

The antibiotic A23187 carries Ca²⁺ across Müller-Rudin membranes made from $\text{1,2-}\text{dierucoyl}\text{-sn-}\text{glycero}\text{-3-}\text{phosphocholine}$ and $\text{n-}\text{decane}$. The conductance of the membranes is not increased by the Ca²⁺-transport. The flux depends linearly on Ca²⁺ concentration and ionophore concentration (above pH 6). It increases with increasing pH, approximately by a factor of 4–5 between pH 6 and pH 8. Maximal Ca²⁺-fluxes of about $\text{10}{}^{\mathrm{?10}}\text{mol}\text{·}\text{cm}{}^{\mathrm{?2}}\text{·}\text{s}{}^{\mathrm{?1}}$ were found. A counter transport of H⁺ could not be detected.The complex formation between A23187 and Ca²⁺ in egg phosphatidylcholine vesicles was studied spectroscopically. The results are consistent with the formation of a 2 : 1 complex. Optical absorption measurements on single phosphatidylcholine membranes were used to calculate the concentration of membrane-bound ionophore A23187.     

Factors affecting the esterification of lipoprotein cholesterol by lecithin: cholesterol acyl transferase.

Lecithin: Cholesterol Acyltransferase (LCAT) esterified relatively small amounts of cholesterol from very low density lipoproteins (VLDL), low density lipoproteins (LDL) or high density lipoproteins (HDL) in the presence of 5% human serum albumin (HSA). On the other hand, in the presence of very high density (>1.225 g/ml) plasma fraction (F-4), the enzyme esterified cholesterol from VLDL at considerably higher rates than from LDL or HDL. VLDL together with some component present in the very high density plasma fraction (F-4) may thus provide a highly efficient complex resulting in a favorable configuration of substrate lipids for the enzyme.     

Melanogenesis in cultures of peripheral nervous tissue: I. The origin and prospective fate of cells giving rise to melanocytes

Chick embryo spinal ganglia, peripheral nerves, and connective tissue usually associated with ganglia were cultured separately using several combinations of media and substrata. Melanocytes appear in cultures of both ganglia and peripheral nerves. The only cell type common to both the ganglion and peripheral nerve that could account for the observed pigment cells was the population of small cells with intensely staining nuclei that normally associates closely with nerve cell bodies and fibers. These cells could be distinguished morphologically from fibroblastic cells, which originated in the connective tissue capsule and did not undergo melanogenesis. We conclude that these small cells are supportive (Schwann, satellite, and perineurial) cell precursors and are one source of melanocytes in cultured peripheral nervous tissue.     

Isolation and analysis of neutral glucans from Ecklonia radiata and Cystophora scalaris

Neutral glucans were isolated from the stipes and fronds of Eklonia radiata and Cystophora scalaris. Partial acid hydrolysis revealed the presence of gentiobiose and laminara-oligosaccharides. Methylation analysis, periodate oxidation, and enzyme studies indicated that the glucans contain β-(1→3) and β-(1→6) linkages. Methylation studies showed that branching in these glucans occurs via a 1,3,6-tri-O- substituted residue with a frequency of one branch point per seven glycosyl residues. In contrast to laminaran from Laminaria digitata, the intrachain (1→3)- and (1→6)- glucopyranoside occur in a molar ratio of 1:1. Enzymic hydrolysis confirmed the absence of long segments of (1→3)-linked residues in the glucans.     

Comparative evaluation of quantum theory of nerve excitation

Deliberate evaluation of the quantum theory of nerve excitation is made by comparing it with Hill's theory in fitting the experimental data on threshold-frequency relation, optimum frequency (v₀) for nerve excitation and strength-duration relation. Decrease of v₀ and increase of all the time constants (Hill's λ andk, Wei'sT ₂ and spike durationw) with decreasing temperature are interpreted on the basis of the dipole relaxation timeT ₂ but inexplicable from Hill's theory or any other existing theory. The closeness ofk,T ₂ andw values is explained. A variety of experimental results obtained by others is discussed. Finally, a comparison is made between the Hodgkin-Huxley equations and the quantum theory. Most of the facts (electrical and non-electrical) tend to support the thesis that nerve excitation is a macroscopic expression of quantum transitions of dipoles between energy states.     

Human Y-chromatin : II. DNA replication

The DNA synthesis of the human Y-chromatin in its various morphological configurations was studied by labeling with tritiated thymidine (³H-TdR) and autoradiography. Continuous terminal and pulse labeling studies revealed that while condensed, the Y-body lagged in DNA synthesis behind the rest of the nucleus. The highest rate of incorporation of ³H-TdR by the Y-body occurred when it was dispersed. These observations are consistent with the replication characteristics of the Y chromosome as determined by conventional late labeling studies and strongly suggest that the Y-body uncoils at the time it replicates its DNA.     

Role of fructose 2,6-bisphosphate in the regulation of glycolysis and gluconeogenesis in chicken liver

Glucagon and dibutyryl cyclic AMP inhibited glucose utilization and lowered fructose 2,6-bisphosphate levels of hepatocytes prepared from fed chickens. Partially purified preparations of chicken liver 6-phosphofructo-1-kinase and fructose 1,6-bisphosphatase were activated and inhibited by fructose 2,6-bisphosphate, respectively. The sensitivities of these enzymes and the changes observed in fructose 2,6-bisphosphate levels are consistent with an important role for this allosteric effector in hormonal regulation of carbohydrate metabolism in chicken liver. In contrast, oleate inhibition of glucose utilization by chicken hepatocytes occurred without change in fructose, 2,6-bisphosphate levels. Likewise, pyruvate inhibition of lactate gluconeogenesis in chicken hepatocytes cannot be explained by changes in fructose 2,6-bisphosphate levels. Exogenous glucose caused a marked increase in fructose 2,6-bisphosphate content of hepatocytes from fasted but not fed birds. Both glucagon and lactate prevented this glucose effect. Fasted chicken hepatocytes responded to lower glucose concentrations than fasted rat hepatocytes, perhaps reflecting the species difference in hexokinase isozymes.     

Proton magnetic resonance studies of lipid bilayer membranes Experimental determination of inter- and intramolecular nuclear relaxation rates in sonicated phosphatidylcholine bilayer vesicles

We have determined the relative magnitudes of the intra- and intermolecular contributions to the nuclear magnetic relaxation rates of the methylene protons of the hydrocarbon chains in phosphatidylcholine bilayer vesicles over a range of temperatures and at two NMR frequencies (100 and 220 MHz). These measurements have been made by the isotopic dilution method using deuterated phosphatidylcholines containing fully deuterated hydrocarbon chains. The results showed that both the methylene linewidths and the spin-lattice relaxation rates are dominated by intramolecular dipolar interactions. Both the intra- and intermolecular contributions to the spin-lattice relaxation rate were found to decrease with increasing temperature and to exhibit a frequency dependence, the rates being higher at the lower NMR frequency in both cases. These observations indicate that both intra- and intermolecular dipolar interactions are modulated by anisotropic motions. In the case of the intermolecular dipolar fields, it is proposed that they are modulated both by the rapid rotational isomerization of the hydrocarbon chains as well as by lateral diffusion of the lipid molecules. That the hydrocarbon chain motion must be fairly effective in effecting efficient spin-lattice relaxation is evident from the negligible intramolecular interchain contribution to the relaxation found in the present work.