Cellular and molecular mechanisms controlling melatonin release by mammalian pineal glands

1. The pineal gland is regulated primarily by photoperiodic information attaining the organ through a multisynaptic pathway initiated in the retina and the retinohypothalamic tract. 2. Norepinephrine (NE) released from superior cervical ganglion (SCG) neurons that provide sympathetic innervation to the pineal acts through alpha1- and beta 1- adrenoceptors to stimulate melatonin synthesis and release. 3. The increase in cyclic AMP mediated by beta 1-adrenergic activation is potentiated by the increase in Ca2+ flux, inositol phospholipid turnover, and prostaglandin and leukotriene synthesis produced by alpha 1-adrenergic activation. 4. Central pinealopetal connections may also participate in pineal control mechanisms; transmitters and modulators in these pathways include several neuropeptides, amino acids such as gamma-aminobutyric acid (GABA) and glutamate, and biogenic amines such as serotonin, acetylcholine, and dopamine. 5. Secondary regulatory signals for pineal secretory activity are several hormones that act on receptors sites on pineal cells or at any stage of the neuronal pinealopetal pathway.     

High and low molecular weight fractions of humic and fulvic acids

Summary The present investigation was initiated to study molecular weight fractionation of humic compounds isolated from surface samples of an Ultisol (Red Yellow Pc lzolic soil) and a Spodosol (Podzol), using fine (mol. wt. cutoff 3500) and coarse (mol. wt. cutoff 12000) dialysis membranes and sephadex gel filtration. Characterization of the humic fractions was conducted by elemental analysis and infrared spectroscopy. The results confirmed that fulvic acid (FA) was higher in ash and elemental content than humic acid (HA). With careful purification the amount of ash was found to be reduced to a minimum, but not to zero. Sephadex gel filtration revealed that fine HA (obtained with fine membranes) was composed of smaller amounts of HA-I (high mol. wt.) and large amounts of HA-II (low mol. wt. fraction). Coarse HA (obtained with coarse membranes) had almost equal amounts of HA-I and HA-II. Fine or coarse FA yielded only low molecular weight components after elution through sephadex. Infrared spectra of the humic fractions were indicative for the presence of phosphoglyceric acid as a possible constituent of the low molecular weight fraction of humic compounds.Contribution of the Univ. of Georgia, Agric. Expt. Sta., College Sta., Athens, Ga. Permission for the publication herein of Sadtler Standard spectrum has been granted, and all rights are reserved by Sadtler Res., Inc.Contribution of the Univ. of Georgia, Agric. Expt. Sta., College Sta., Athens, Ga. Permission for the publication herein of Sadtler Standard spectrum has been granted, and all rights are reserved by Sadtler Res., Inc.     

Electrophoretic subfractionation of low and high molecular weight humic acids fractions

Summary Soil humic acid was fractionated on a molecular weight basis either using Sephadex gel filtration or electrophoresis on a discontinuous polyacrylamide gel. Low and high molecular weight fractions obtained by these two methods were choosen for subsequent subfractionation using electrophoretic methods. The high and low molecular weight fractions yielded several subfractions after separation by isotachophoresis or isoelectric focusing. Components of the high molecular weight fractions occupied the upper portion of the mobility train; components of the low molecular weight fractions lead the mobility train. Adsorption by Sephadex was avoided by using 4M urea as an eluent. The elution of the humic substances adsorbed to the polyacrylamide gel matrix was achieved by using a 0.1M Tris –0.025M EDTA solution.     

Effect of light and darkness on pineal hydroxyindole-O-methyl transferase (HIOMT) in Japanese quail

The optimal incubation temperature for assay of the melatonin-forming enzyme, hydroxyindole-O-methyl transferase (HIOMT), of the Japanese quail pineal, was determined to be 47° C. In adult quail, HIOMT activity was high in continuous light and in the light phase of $\text{12}\text{L}\text{12}\text{D}\text{and}\text{12}\text{D}\text{12}\text{L}$, and was low in continuous darkness and in the dark phase of $\text{12}\text{L}\text{12}\text{D}\text{and}\text{12}\text{D}\text{12}\text{L}$. Age of the bird and length of the lighting treatment seemed to be important factors in demonstrating this effect of light and darkness on HIOMT activity.     

Hydroxyindole-O-methyl transferase (HIOMT) and serotonin in the pineal gland of the vizcacha (Lagostomus maximus maximus)

Pineal glands of vizcachas collected from their natural habitat were studied. Hydroxyindole-O-methyl transferase (HIOMT) activity showed a great individual dispersion. However, the enzyme activity was significantly higher in males than in females. Serotonin was also assayed in the glands. It did not show significant differences between both sexes. The results are discussed in relation to the natural life conditions of this rodent.     

Inhibition of the p53-hdm2 interaction with low molecular weight compounds

The hdm2 protein, upon binding to p53, inhibits its tumor suppressor activity. The inhibition of the p53-hdm2 interaction represents therefore a new therapeutic strategy to activate wild type p53 in tumors. Potent low molecular weight compounds inhibiting this protein-protein interaction, which are active in vivo, have just been identified. This offers new perspectives and hopes in this research area.     

Inhibition of platelet aggregation by dinitrosyl iron complexes with low molecular weight ligands

The dinitrosyl iron complexes (DNIC) with thiosulphate, cysteine or phosphate were shown to inhibit in vitro (in citrate plasma) the human platelet aggregation induced by ADP, collagen or adrenaline. This effect cannot be explained by the toxic action of DNIC on the platelet membrane, since DNIC-pretreated platelets are capable of aggregating under the action of 10(-8) M/ml of phorbol ester, which is known to cause direct activation of protein kinase C. The antiaggregatory activity of DNIC exceeds that of Na-nitroprusside and seems to be due to nitric oxide capable to activate guanylate cyclase of platelets. Using the EPR method, it was shown that addition of DNIC to platelet-enriched plasma results in a rapid transfer of Fe(NO)2 groups to the coupled RS(-)-groups proteins of plasma and, apparently, of platelet membrane proteins. These protein DNIC seem to be the source of NO which inhibits human platelet aggregation.     

Anterograde tracing of retinal axons in the avian embryo with low molecular weight derivatives of biotin

Several reactive biotin esters were injected into the eyes of chick and quail embryos at various stages of development. Four of the biotin esters reacted with molecules of the eye tissue and were detected with light and electron microscopy in fluorescein isothiocyanate and peroxidase-avidin incubated sections and whole mounts. Intra and extracellular components of the lens, the vitreous body, and the retina were labeled to different degrees. Three of the biotin esters (biotin-N-hydroxysuccinimidester, biotin-epsilon-aminocaproic acid-N-hydroxysuccinimidester, and desthiobiotin-N-hydroxysuccinimidester) prominently marked the optic fiber layer in the retina and the biotin labels were transported along the optic pathway. The tracers were detected up to the growth cone of axons 24 to 36 hr after injection. Explants from biotin marked retinas were cultured on collagen or basal laminae. During culturing axons grew out from these explants into the substratum showing that labeled tissue and nerve fibers were viable. The development of the optic pathway at the chiasma of quail embryos was studied using the biotin/avidin tracing. The bulk of fibers emerging from the retina crossed as shown by double labeling of both optic nerves in a complex pattern of segregated and interdigitizing axon bundles at the chiasma toward the contralateral side of the brain. From stage 25 onward a minor ipsilateral projection was found. At the same developmental stage a few fibers traveled into the contralateral optic nerve and grew retrogradely toward the contralateral eye. The percentage of specimens having this retino-retinal projection increased during development from 53% (stage 24 to 27; E3.5-E5.5) to 89% (stage 29 to 35; E6-E8) and declined to 40% at late embryogenesis (stage 37 to 41; E9-E12). The fact that all retinal axons were found within predictable pathways with some of them running in the wrong direction suggests that nerve fiber pathways provide accurate positional information, but at best weak directional information for growing nerve fibers.     

Association of c-myc protein with enzymes of DNA replication in high molecular weight fractions from mammalian cells.

Oncoprotein c-myc is expressed in proliferating but not quiescent mammalian cells, and its overexpression or inappropriate expression is associated with malignant transformation. However, in spite of an intense interest, the normal function of this protein has remained elusive. As a step towards the elucidation of the function of c-myc protein, we studied its distribution within several types of cells, including HL 60, K 562, COLO 320, and CHEF/18 cells. In all of the cells studied, c-myc protein was detected in high molecular weight protein fractions, in 350-600 Kd range, in gel-exclusion chromatography and sucrose gradient centrifugation. This distribution of c-myc protein coincided with the distribution of DNA polymerase alpha and several other enzymes necessary for DNA replication. The data suggest that c-myc product may be a component of the replitase complex of enzymes involved in nuclear DNA replication.     

Kinetics of radiolabelled (99mTc) heparin and low molecular weight heparin fractions CY 216, CY 222 in patients with uncomplicated myocardial infarction

Kinetics of plasmatic disappearance of radiolabelled (99mTc) heparin (Choay, Mr mean 15,000) and low molecular weight heparin (LMWH) fraction CY 216 (Choay, Mr mean 5,000) and CY 222 (Choay, Mr mean 4,000) was compared in 2 women and 8 men (aged 50-71, mean 65 years) with uncomplicated myocardial infarction. The three technetiated heparins were consecutively injected intravenously (67 nanomoles) to each of 10 patients, at intervals of 3-5 days, 14-28 days after acute cardiac onset. The plasma radioactivity was counted in blood samples collected within a period of 5 h. Radiolabelled heparin and LMWH fractions CY 216, CY 222 disappeared from plasma following a biexponential clearance curve with a fast and slow component reflecting the biodistribution (alpha) and elimination (beta) phase. The bioavailability values (AUC, t0.5 alpha, t0.5 beta) as well as distribution and elimination rates were similar for all three technetiated heparins. The bulk of injected 99mTc-heparin or LMWH fraction was rapidly distributed to the tissular compartment (t0.5 alpha = 13 min), whereas the radiocomplex remaining in the circulation was slowly eliminated with a half-time (t0.5 beta) of an average 320 min. Radioactivity eliminated from plasma was only partially (30-50%) excreted in the urine. The results indicate that after a low-dose intravenous administration LMWH fractions CY 216 and CY 222 maintain the pharmacokinetics properties of standard heparin, especially the rapid distribution to the tissular compartment.     

Inhibition studies with rationally designed inhibitors of the human low molecular weight protein tyrosine phosphatase

The human low molecular weight protein tyrosine phosphatase (HCPTP) is ubiquitously expressed as two isoforms in a wide range of human cells and may be involved in regulating the metastatic nature of epithelial tumors. A homology model is presented for the HCPTP-B isoform based on known X-ray crystal structures of other low molecular weight PTPs. A comparison of the two isoform structures indicates the possibility of developing isoform-specific inhibitors of HCPTP. Molecular dynamics simulations with CHARMM have been used to study the binding modes of the known adenine effector and phosphate in the active site of both isoforms. This analysis led to the design of the initial lead compound, based on an azaindole ring moiety, which was then also evaluated computationally. A comparison of these simulations indicates the need for a phosphonate group on the indole and provides insight into inhibitor binding modes. Compounds with varying degrees of structural similarity to the azaindole have been synthesized and tested for inhibition with each isoform. These molecular systems were examined with the program AutoDock, and comparisons made with the kinetics and the explicit simulations to validate AutoDock as a screening tool for potential inhibitors. Two compounds were experimentally found to have sub-millimolar inhibition, but the greater solubility of one reinforces the need for experimental testing alongside computational analysis.     

Effect of low molecular weight heparin and different heparin molecular weight fractions on the activity of the matrix-degrading enzyme aggrecanase: structure-function relationship

The matrix-degrading enzyme aggrecanase has been identified in cartilage and is largely responsible for cartilage breakdown. The present study determined the efficacy of different heparin molecular weight fractions (HMWFs) and low molecular weight heparins (LMWHs) on aggrecanase activity. Aggrecanase activity was determined using biotinylated peptide substrate, which was immobilized onto streptavidin-coated 96-well plates; aggrecanase enzyme was then added. Proteolysis of the substrate at the specific amide bond was detected using specific antibody for the neoepitope generated. HMWFs ranging from 1,700 to 12,000 Da demonstrated a concentration-dependent inhibitory efficacy of aggrecanase activity, with a Ki ranging from 5,000 nM down to 1 nM as a function of the molecular weight. The higher the molecular weight distribution, the greater the inhibitory efficacy of the heparin fragments toward aggrecanase activity. The absence or presence of antithrombin did not alter the affinity of heparin in inhibiting aggrecanase. Additionally, tissue factor pathway inhibitor at various levels did not alter the activity of aggrecanase. LMWHs demonstrated different levels of potency in inhibiting aggrecanase activity as a function of their average molecular weight distribution. Tinzaparin (average molecular weight = 6,500 Da) and enoxaparin (average molecular weight = 4,500 Da) demonstrated a Ki of 20 and 80 nM, respectively. The aggrecanase inhibitory effect of LMWH might contribute to blocking inflammation and tumor invasion by inhibiting aggrecanase activity and maintaining an intact extracellular matrix barrier.     

Steady-state kinetics of low molecular weight (type-II) NADH dehydrogenase.

(1) The steady-state kinetics of the NADH dehydrogenase activity of Type-II (low molecular weight) NADH dehydrogenase with the acceptors ferricyanide, cytochrome c and 2,6-dichloroindophenol are consistent with the simultaneous operation of an ordered and a ping-pong mechanism. Thus, depending on the acceptor concentration, the reduced enzyme is preferentially oxidized before or after NAD+ disociates from it. (2) The acceptors are able to oxidize the reduced enzyme and its NAD+ complex equally well. In contrast to the kinetics of the Type-I (high molecular weight) enzyme, double substrate inhibition is not found, implying that the site of oxidation of the reduced enzyme by acceptors and the NADH-binding site are remote. (3) With the indophenol, in the concentration range measured, the ordered mechanism is mainly operative. At infinite NADH and acceptor concentrations the rate constant of the reduction of enzyme by bound NADH is measured. (4) With ferricyanide and cytochrome c, in the concentration range measured, erroneous conclusions may be drawn from extrapolations owing to the fact that extrapolated lines in double-reciprocal plots of turnover number against acceptor concentration, at different NADH concentrations, intersect in the third quadrant. A method is described that allows the extrapolation of these data to zero acceptor concentrations. (5) The relation between activity and NADH concentration is sigmoidal (h = 2.0) with ferricyanide or cytochrome c as acceptor, but hyperbolic with 2,6-dichloroindophenol. The latter is also an inhibitor, competitive with respect to NADH. It is concluded that this two-electron acceptor, like ubiquinone, acts as an allosteric effector. (6) Type II is isolated from Type I without gross changes in tertiary structure, as judged by the unaltered rate constants of dissociation of NADH (k-1) and NAD+ (k4) and association of NADH (k1). (7) Type II differs from Type I in two respects, (a) The accessibility of the acceptors is greater by at least two orders of magnitude (k3). (b) The redox potential of the prosthetic group FMN is 120 mV less, as judged by a drop in the value of k2 by four orders of magnitude. It is suggested that one or more of the iron-sulphur proteins present in Type-I but lacking in Type-II dehydrogenase functions as an effector, regulating the redox potential of the FMN.