Helix formation in reduced,S-carboxymethylated human choriogonadotropin β subunit and tryptic peptides

The β subunit of human choriogonadotropin (hCGβ) and its asialoderivative were digested with trypsin and then reduced and S-carboxymethylated. A series of peptides were purified which corresponded to residues 1–43, 44–95, 96–114, and 123–145 of the 145 amino acid residue glycoprotein. The two N-linked oligosaccharides were present on the amino terminal peptide, and three of the four O-linked oligosaccharides were present on the carboxy terminal peptide. Circular dichroic spectra between 190–240 nm were obtained on reduced, S-carboxymethylated (RCM) hCGβ and the above peptides, both in aqueous solution and in the helicogenic solvent 80% (vol/vol) trifluoroethanol (TFE). In aqueous solution there was evidence of only limited helicity in the peptides and RCM-hCGβ however, in the presence of TFE, peptides 1–43 and 44–95 exhibited significant helicity, as did the full-length linear chain. The helicity developed in TFE by RCM-hCGβ appears much greater than that which occurs in the native, disulfide-intact form, thus suggesting that the disulfides prevent expression of helicity in regions with α-helix potential. Application of the Chou-Fasman secondary structure predictive algorithm to hCGβ suggested that several regions of helix potential, in particular regions 14–21, 59–69, and perhaps 80–88, may account for much of the helicity observed in peptides 1–43 and 44–95, respectively, in TFE. The region from 96–145 has no significant potential for helicity, consistent with the measured circular dichroic spectra of peptides 96–114 and 123–145. These results demonstrate that helicity can occur in the linear form of hCGβ, and this secondary structure can best be attributed to the amino terminal and the middle portion of the molecule. Several potential regions of β-structure and β-turns were also suggested.     

Short-term action of insulin on Aplysia neurons: Generation of a possible novel modulator of ion channels

In mollusks as in other animals, peptides can act as hormones, growth factors, and neurotransmitters. The presence of insulin in vertebrate brain as well as its actions on nerve cells led us to examine the electrophysiological effects of the mammalian hormone on Aplysia neurons. Application of insulin extracellularly causes hyperpolarization of L14 and L10, identified neurons of the abdominal ganglion. This hyperpolarization is associated with a decreased membrane conductance that reverses at ?35 mV. We also injected inositol phosphate glycan (IPG) into the identified neurons. This complex sugar, which was purified from rat liver and which is a putative second messenger for insulin in nonneural vertebrate cells (Saltiel and Cuatrecasas, 1986; Saltiel, Osterman, and Darnell, 1988), causes hyperpolarization with decreased membrane conductance in L14 and L10 similar to the effects of insulin. Furthermore, exposure of isolated ganglia to insulin results in the generation of IPG with a compensating decrease in its glycosyl-phosphatidylinositol precursor. We suggest that, in addition to its other roles, insulin may function as a neuropeptide transmitter using IPG as a second messenger.     

The inhibitor reacting with a tumour cell surface protease can be exchanged with plasminogen activator inhibitor (PAI-1)

Tumour cells possess the cell surface protease guanidinobenzoatase (GB) which can be located by the fluorescent probe 9-amino acridine (9-AA). Frozen sections and formaldehyde fixed sections of tumour tissue were used to demonstrate the interactions between GB, 9-AA and two protein inhibitors of GB. A cytoplasmic extract from the tumour tissue, and a purified inhibitor of plasminogen activator (PAI-1) were shown to be exchangeable components of the enzyme-inhibitor complex on the fixed tumour cell surfaces. The evidence suggests that GB is functionally very similar to plasminogen activator and that this enzyme can be regulated by protein inhibitors in vivo and also by changes in the redox potential at the cell surface.     

Standing biomass and production in water drainages of the foothills of the Philip Smith Mountains, Alaska

In the foothills of the Philip Smith Mountains, Brooks Range, Alaska, tussock tundra is the most widely distributed vegetation, and it occurs on rolling hills and in valleys that were shaped by a sequence of Pleistocene glaciations. In this study, aboveground standing biomass and production were compared in "intertrack tundra" areas that were relatively homogenous with respect to downslope drainage and adjacent "water tracks" that acted to channel water flow to the valley bottom stream. Comparisons of biomass, leaf area index, and specific leaf weight were also made between upper and lower slope positions. Similarities and differences of vegetation structure are examined with respect to graminoid, deciduous shrub, evergreen shrub, herbaceous, and bryophyte components.
Water tracks were found to have 1.5–1.7 times the biomass of intertrack tundra, and production (excluding secondary growth) in water tracks was 40% greater than in intertrack tundra. The aboveground biomass for all areas studied and the annual production values were similar to those found in other studies of tussock tundra. While only slight differences in depth of thaw occurred in water tracks and intertrack tundra during June and early July, water tracks thawed more deeply with the onset of summer rains. Warmer temperatures at 40 cm depth in July and August may have increased nutrient availability, whereas greater rooting depth and movement of water may have increased nutrient capture in water tracks as compared with the intertrack areas. Greater biomass and a deeper thaw depth occurred at upper slope locations.     

Role of the Blood-Brain Barrier in the Formation of Long-Chain ω-3 and ω-6 Fatty Acids from Essential Fatty Acid Precursors

Elongated, more highly polyunsaturated derivatives of linoleic acid (18:2 omega-6) and linolenic acid (18:3 omega-3) accumulate in brain, but their sites of synthesis and mechanism of entry are not well characterized. To investigate the role of the blood-brain barrier in this process, cultured murine cerebromicrovascular endothelia were incubated with [1-14C]18:2 omega-6 or [1-14C]18:3 omega-3 and their elongation/desaturation products determined. The major metabolite of 18:2 omega-6 was 20:4 omega-6, whereas the primary product from 18:3 omega-3 was 20:5 omega-3. Although these products were found primarily in cell lipids, they were also released from the cells and gradually accumulated in the extracellular fluid. Eicosanoid production was observed from the 20:4 omega-6 and 20:5 omega-3 that were formed. No 22:5 omega-6 or 22:6 omega-3 fatty acids were detected, suggesting that these endothelial cells are not the site of the final desaturation step. Although the uptake of 18:3 omega-3 and 18:2 omega-6 was nearly identical, 18:3 omega-3 was more extensively elongated and desaturated. Competition experiments demonstrated a preference for 18:3 omega-3 by the elongation/desaturation pathway. These findings suggest that the blood-brain barrier can play an important role in the elongation and desaturation of omega-3 and omega-6 essential fatty acids during their transfer from the circulation into the brain.     

Endogenous Benzodiazepine Receptor Ligands in Human and Animal Hepatic Encephalopathy

The role of endogenous benzodiazepine receptor ligands in the pathogenesis of hepatic encephalopathy was studied in humans and in rat models of hepatic encephalopathy. Endogenous benzodiazepine ligands were extracted from rat brain and human CSF by acid treatment and purification by HPLC. Detection and partial characterization of these endogenous benzodiazepine ligands were carried out using both radioreceptor binding assays and radioimmunoassays with anti-benzodiazepine antibodies. Four different benzodiazepine receptor ligands were identified in human and rat tissue, two of which may be diazepam and desmethyldiazepam, based on elution profiles and anti-benzo-diazepine antibody reactivity. Human CSF and serum from patients with hepatic encephalopathy contained approximately 10 times more endogenous benzodiazepine receptor ligand than CSF from controls or nonencephalopathic patients with liver disease. The levels of brain benzodiazepine receptor ligand compounds were also increased approximately 10-fold in rats suffering from fulminant hepatic failure, but not in rats with portacaval shunts, a model of chronic hepatic disease. The increased concentrations of these substances could be behaviorally significant and may contribute to the pathogenesis of hepatic encephalopathy.     

Intrasynaptosomal Sequestration of [3H]Amphetamine and [3H]Methylenedioxyamphetamine: Characterization Suggests the Presence of a Factor Responsible for Maintaining Sequestration

We examined the incorporation of [3H]methylenedioxyamphetamine ([3H]MDA) and [3H]amphetamine into rat brain synaptosomes. Saturation studies, using increasing concentrations of nonradioactive ligand, revealed that [3H]-MDA interacted with two saturable sites that were sensitive to boiling of the tissue. Eadee-Scatchard plots of [3H]MDA saturation data were curvilinear; nonlinear curve-fitting analysis of these data suggested the presence of high- and low-affinity [3H]MDA sites of association: KD high = 295 nM, Bmax high = 32 pmol/mg of protein; KD low = 45 microM, Bmax low = 5.2 nmol/mg of protein. Association of [3H]MDA to the low-affinity site was dependent on the presence of isotonic sucrose in the incubation medium. The high capacities of these sites argue against a bimolecular interaction of [3H]MDA with monovalent protein binding sites. [3H]MDA incorporation was reduced under conditions that disrupt the integrity of plasma membranes, such as sonication, incubation in hypotonic media, and incubation in the presence of the detergent digitonin. These data indicate that [3H]MDA incorporation into synaptosomes may represent an internalization and sequestration phenomenon. [3H]MDA incorporation was also reduced by preincubation of the synaptosomal preparation at 37 degrees C or in hypotonic buffer at 4 degrees C, a result suggesting that this sequestration is maintained by an intrasynaptosomal component that is lost under the preincubation conditions described above. [3H]MDA incorporation was pH dependent (maximal at pH 7.5) and temperature sensitive (maximal incorporation occurred at 21 degrees C and was substantially reduced at 37 degrees C). [3H]Amphetamine was also incorporated into synaptosomes, and this incorporation was sensitive to the same physical manipulations of the tissue preparation as [3H]MDA incorporation. The synaptosomal sequestration of both [3H]MDA and [3H]amphetamine was inhibited by permeant cations, such as sodium and potassium, a result suggesting that the proposed intrasynaptosomal component that maintains the sequestration is anionic. Preliminary pharmacological profiles of [3H]MDA and [3H]amphetamine sequestration were identical. The rank order of inhibitor potencies for the incorporation of both ligands was desipramine greater than amphetamine greater than MDA greater than methylphenidate. This order of potency does not correspond to the lipophilicity of the test drugs. The synaptosomal incorporation and sequestration of [3H]MDA, [3H]methylenedioxymethamphetamine, and [3H]amphetamine described in the present report may be important in the molecular mechanism of action of monoamine release induced by the amphetamines.     

Subacute and Chronic In Vivo Lithium Treatment Inhibits Agonist- and Sodium Fluoride-Stimulated Inositol Phosphate Production in Rat Cortex

We have investigated the effects of in vivo lithium treatment on cerebral inositol phospholipid metabolism. Twice-daily treatment of rats with LiCl (3 mEq/kg) for 3 or 16 days resulted in a 25-40% reduction in agonist-stimulated inositol phosphate production, compared with NaCl-treated controls, in cortical slices prelabelled with [3H]inositol. A small effect was also seen with 5-hydroxytryptamine (5-HT) 24 h after a single dose of LiCl (10 mEq/kg). Dose-response curves to carbachol and 5-HT showed that lithium treatment reduced the maximal agonist response without altering the EC50 value. This inhibition was not affected by the concentration of LiCl in the assay buffer. Stimulation of inositol phosphate formation by 10 mM NaF in membranes prepared from cortex of 3-day lithium-treated rats was also inhibited, by 35% compared with NaCl-treated controls. Lithium treatment did not alter the kinetic profile of inositol polyphosphate formation in cortical slices stimulated with carbachol. Muscarinic cholinergic and 5-HT2 bindings were unaltered by lithium, as was cortical phospholipase C activity and isoproterenol-stimulated cyclic AMP formation. [3H]Inositol labelling of phosphatidylinositol 4,5-bisphosphate was significantly enhanced by 3-day lithium treatment. The results, therefore, indicate that subacute or chronic in vivo lithium treatment reduces agonist-stimulated inositol phospholipid metabolism in cerebral cortex; this persistent inhibition appears to be at the level of G-protein-phospholipase C coupling.     

Modification of Gs-Stimulated Adenylate Cyclase in Brain Membranes by Low pH Pretreatment: Correlation with Altered Guanine Nucleotide Exchange

Pretreatment of rat brain membranes at pH 4.5 before assay at pH 7.4 modifies the function of GTP-binding proteins (G-proteins) by eliminating Gs-stimulated adenylate cyclase activity while increasing opiate-inhibited adenylate cyclase activity. To help characterize the molecular nature of the low pH effect, we labeled Gs and Gi alpha-subunits in both control and low pH-pretreated membranes with the GTP photoaffinity analog [32P]P3 (4-azidoanilido)-P1-5'-GTP ([32P]AAGTP). When membranes were preincubated with unlabeled AAGTP, a persistent inhibitory state of adenylate cyclase was produced, which was overcome in untreated membranes with high (greater than 1 microM) concentrations of guanylyl-5'-imidodiphosphate [Gpp(NH)p]. In low pH-pretreated membranes, this inhibition could not be overcome, and stimulation by Gpp(NH)p was eliminated. Maximal inhibition of adenylate cyclase achieved by incubation with AAGTP was not altered by low pH pretreatment. Incorporation of [32P]AAGTP into Gs (42 kilodaltons) or Gi/o (40 kilodaltons) was unaffected by low pH pretreatment; however, transfer of 32P from Gi/o to Gs, which occurs with low (10 nM) concentrations of Gpp(NH)p in untreated membranes, was severely retarded in low pH-pretreated membranes. Both the potency and efficacy of Gpp(NH)p in producing exchange of [32P]AAGTP from Gi/o to Gs were markedly reduced by low pH pretreatment. These results correlate the loss of Gs-stimulated adenylate cyclase with a loss of transfer of nucleotide from Gi/o to Gs alpha-subunits and suggest that the nucleotide exchange participates in the modulation of neuronal adenylate cyclase.