Isolation and primary structure of novel neurointermediate pituitary peptides derived from the C-terminal of the rat vasopressin-neurophysin precursor (propressophysin)

Four novel peptides were isolated from rat neurointermediate lobes by gel filtration and high-pressure liquid chromatography. Analyses of amino acid composition and sequence showed that all four peptides were derived from the C-terminal portion of propressophysin (CPP); they were identified as the glycopeptides CPP 1-19, CPP 1-20, CPP 22-37 and CPP 22-39. Processing of CPP 1-39 could thus produce the four isolated peptides by specific post-Arg or post-Leu cleavages.     

C-terminal fragments of ACTH stimulate feeding in fasted rats.

Peptides derived from pro-opiomelanocortin, including alpha-MSH and ACTH, play important roles in the regulation of feeding. We investigated the central effect of ACTH 1-39 (ACTH) and peptides derived from the N-terminus (ACTH 1-10, Acetyl-ACTH 1-13-amide [alpha-MSH]) and C-terminus (ACTH 18-39 and ACTH 22-39) of this peptide on feeding in 16 hour-fasted or rats fed ad libitum. As expected, ACTH reduced feeding in fed and previously fasted rats, although this anorectic effect was more pronounced in fasted rats. The N-terminal-derived peptide alpha-MSH, but not ACTH 1-10, reduced cumulative food intake over 2 h after its injection intracerebroventricularly (icv) in 16 h-fasted, but not in fed rats. In contrast, the C-terminal fragments produced a long-lasting increase in feeding in fasted, but not in fed rats. The anorectic effects of N-terminal fragments of ACTH are recognised to be mediated via melanocortin MC4 receptors. However, the orexigenic effects of the C-terminal fragments do not appear to be conducted via MC4 receptors, since neither ACTH 18-39 nor ACTH 22-39 stimulated cAMP accumulation nor inhibited the ACTH-stimulated cAMP accumulation in HEK-293 cells transfected with the recombinant MC4 receptor.     

Response of the hypothalamo-neurohypophyseal axis (AVP system) and the kidney to salt load in young propylthiouracil-treated rats

The development of the ability of the kidney to concentrate urine was studied in normal and propylthiouracil-treated rats by measuring urinary sodium concentration and osmolarity at different ages. It was shown that the normal animals, but not the PTU-treated ones were able to concentrate urine at 35 days of age. The response of the hypothalamo-neurohypophyseal axis (AVP system) and that of the kidney were investigated in the two categories of animals at 35 days of age by measuring concomitantly the AVP content of the hypothalamus and neurohypophysis, the AVP plasma concentration and the natremia and plasma osmolarity at various intervals after intraperitoneal injection of a 5% NaCl solution. In normal, as well as in PTU-treated rats, salt load did not lead to significant modifications of the hypothalamic AVP content. In comparison with normal rats, the neurohypophysis of the PTU-treated ones released AVP more slowly, but with a similar amplitude. In normal rats, the plasma AVP concentration was already maximal 30 min after salt load and then decreased and returned to the normal value at 2 hrs; at the same time, the natremia and plasma osmolarity also recovered their normal value. In contrast, the plasma AVP concentration as well as the natremia remained high 1 h 30 after salt load in PTU-treated rats. It is concluded that PTU-treated young rat is unable to compensate the salt load in normal delays. Beside the slowing in AVP release by neurohypophysis and decreased AVP plasma level, the responsiveness of the kidney seems to be altered.     

Identification and structural analysis of the antimicrobial domain in hipposin, a 51-mer antimicrobial peptide isolated from Atlantic halibut

Hipposin is a potent 51-mer antimicrobial peptide (AMP) from Atlantic halibut with sequence similarity to parasin (19-mer catfish AMP), buforin I (39-mer toad AMP), and buforin II (an active 21-mer fragment of buforin I), suggesting that the antimicrobial activity of these peptides might all be due to a common antimicrobial sequence motif. In order to identify the putative sequence motif, the antimicrobial activity of hipposin fragments against 20 different bacteria was compared to the activity of hipposin, parasin and buforin II. Neither parasin nor the 19-mer parasin-like fragment HIP(1-19) (differs from parasin in only three residues) that is derived from the N-terminal part (residues 1-19) of hipposin had marked antimicrobial activity. In contrast, the fragment HIP(16-36) (identical to buforin II) that is derived from the middle part of hipposin (residues 16-36) had such activity, indicating that this part of hipposin contained an antimicrobial sequence motif. The activity was enhanced when the parasin-like N-terminal sequence was also present, as the fragment HIP(1-36) which consists of residues 1-36 in hipposin was more potent than HIP(16-36). Extending HIP(1-36) with three C-terminal residues-thereby constructing the buforin I-like peptide HIP(1-39) (differs from buforin I in only three residues)-increased the activity further. Also, the presence of the C-terminal part of hipposin (residues 40-51) increased the activity, as hipposin was clearly the most potent of all the peptides that were tested. Circular dichroism structural analysis of the peptides revealed that they were all non-structured in aqueous solution. However, trifluoroethanol and the membrane-mimicking entities dodecylphosphocholine micelles and negatively charged liposomes induced (amphiphilic) alpha-helical structuring in hipposin. Judging from the structuring of the individual fragments, the tendency for alpha-helical structuring appeared to be greater in the C-terminal and the buforin II-like middle region of hipposin than in the parasin-like N-terminal region.     

Distribution of 3H-thymidine in the postnatal hypophysis of the C57BL mouse

The distribution of cells labelled with 3H-thymidine was determined autoradiographically in the adenohypophysis and neurohypophysis of the C57BL mouse during postnatal phases ranging from the newborn to 24 days of age, as well as in the adult. In the newborn, labelled cells are scarce in the neurohypophysis but common in the adenohypophysis. The neurohypophysis shows a surge in labelling at 5-9 days, with a sharp decline thereafter. In the adenohypophysis, labelled nuclei are scarce in the pars tuberalis after 19 days, whereas the pars intermedia and pars distalis continue to show labelled cells. In the pars distalis, at all phases, label occurs in the marginal cells along the hypophysial cleft as well as in deeper-lying cells representing follicular cells. In the adult, follicular cells are more commonly labelled relative to other cells of the hypophysis.     

Effects of colchicine on axonal transport and ultrastructure of the hypothalamo-neurohypophyseal system of the rat

Summary The effect of colchicine on the transport of proteins in the hypothalamo-neurohypophyseal tract of the rat was studied after injection of (³⁵S) cysteine into the supraoptic nucleus (SON) region. Colchicine, dissolved in distilled water and administered subarachnoidally, inhibited the axonal transport of labelled proteins into the neurohypophysis: the radioactivity that was recovered in neurohypophyseal TCA precipitable material was markedly decreased and hardly any radioactivity was found in the neurohypophyseal proteins which were separated by polyacrylamide gel disc electrophoresis.As revealed by electron microscopy the SON cell bodies showed marked changes after treatment with colchicine: a deeply folded nucleolemma; a pronounced, granular nucleolus; a dispersed chromatin; a zonal distribution of cell organelles with mitochondria and lysosomes accumulated at the periphery, crowded ribosomes, often arranged as polyribosomes and richly branching short profiles of endoplasmic reticulum filled with filamentous material forming an inner perinuclear zone separated by enlarged Golgi complexes.The profiles of elongated Herring bodies in the infundibulum were increased. The axon terminals were filled with heavily osmiophilic neurosecretory granules. The neurofilaments were slightly or moderately increased in number. No apparent changes were observed with regard to the neurotubuli in the SON neurons. The glial cells of the supraopticoneurohypophyseal tract showed reactive changes with a proliferation of filamentous elements. The biochemical and ultrastructural findings are discussed especially with respect to the mechanisms of transport and release of neurosecretory granules.     

Production of a specific antiserum by synthetic C-terminal fragment of glucagon

Seven rabbits were immunized with a synthetic C-terminal glucagon fragment [15--29] conjugated with bovine serum albumin by means of glutaraldehyde. Antisera for glucagon were produced in all the animals after six injections of the conjugate. One of them revealed a higher titer antiserum (G42), which did not cross react with gut glucagon-like immunoreactive material, secretin, insulin, gastric inhibitory polypeptide or vasoactive intestinal peptide. From the results of inhibition of 125 I-glucagon in binding with the antiserum by various glucagon-related fragments the immunogenic determinant of the antiserum was proved to be in the C-terminal residue of the glucagon molecule, although peptide [17--29] or [21--29] reacted weakly with the antiserum. The plasma glucagon levels measured by antiserum G 42 during an arginine test in five normal subjects were superposed on those obtained by other antiserum (G21), specific for pancreatic glucagon. Furthermore, a comparable standard curve for glucagon was obtained using antiserum G42, when a labelled p-hydroxyphenylacetylated glucagon fragment [15--29] was employed as a tracer. The present study clearly demonstrated that the C-terminal glucagon fragment could yield a specific antiserum for pancreatic glucagon, supporting the proposal that the C-terminal fragment of glucagon is responsible for such specific antisera. Furthermore, it is concluded that immunoassay for glucagon could be performed using the labelled glucagon fragment as a tracer.     

Use of substance P fragments to differentiate substance P receptors of different tissues

The C- and N-terminal fragments of substance P were compared to the parent molecule with respect to their ability to: (a) contract the isolated guinea pig ileum, (b) induce salivation in the rat, (c) excite single cat dorsal horn neurones, and (d) induce scratching by intracranial injections in mice. C-terminal fragments as small as the heptapeptide were potent SP agonists on all assay systems. C-terminal fragments containing five amino acids or less were, at most, only weakly active. The C-terminal hexapeptide was a potent SP receptor stimulant on the isolated guinea pig ileum and, when directly applied by microiontophoresis, on cat dorsal horn neurons. However, the same compound was only 2-5% as potent as substance P in eliciting salivation and scratching in vivo, an indication that this fragment may be especially labile to enzymatic degradation. N-terminal fragments were totally inactive on the isolated guinea pig ileum. On the rat salivation and central nervous system assays, however, N-terminal fragments were capable of weak SP-like activity. It is concluded that SP receptors exist in multiple forms which we have labelled SP1 and SP2 receptors for those insensitive or sensitive to N-terminal fragments, respectively.     

Purification of twelve cyanogen bromide fragments from bovine plasma fibronectin and the amino acid sequence of eight of them. Overlap evidence aligning two plasmic fragments, internal homology in gelatin-binding region and phosphorylation site near C terminus

Twelve cyanogen bromide fragments (CB1-12) from bovine plasma fibronectin have been isolated and eight of these completely sequenced. Altogether they account for 502 of the total expected 1880 residues in each of the two chains of fibronectin. Four of these fragments (CB1-4) constitute residues 1-289 in fibronectin with CB4 overlapping the N-terminal 29-kDa plasmic fragment to the second plasmic fragment, of 170-kDa in fibronectin. Fragments CB 5-9 are all contained within a 45-kDa gelatin-binding region, which is N-terminal in the 170-kDa fragment. The sequence of two of these five fragments in the 45-kDa fragment (CB7-8) contains two mutually homologous stretches with 57% sequence identity. Another two fragments (CB10-11) are derived from the heparin-binding region of the 170-kDa fragment. CB12 constitutes the C-terminal 13-residue stretch in fibronectin and contains a partly phosphorylated serine residue in the C-terminal sequence: -Arg-Glu-Asp-Ser(P)-Arg-Glu.     

Light microscope radioautographic study of glycoprotein secretion in the hypothalamic-neurohypophysial system of the rat,after L-fucose-3H injection

Summary Fucose-³H was injected into the cerebral ventricle of rats that were killed at several time intervals after injection. Semi-thin sections of the supraoptic nucleus and neurohypophysis were processed for radioautography and analysed quantitatively. Silver grains indicating the site of fucose-labeled glycoproteins were first located at the perinuclear region of the secretory neurons. The highest silver-grain density in these cells was observed at 2 h after injection, declining afterwards. Silver grains over the neurohypophysis were observed from 2 h on, reached a peak at 1 day after injection and decreased in the subsequent time intervals. The distributions of the silver grains over the neurohypophysis fitted Poissonian distributions and these were shown to be heterogeneous at the several time intervals. Pituicytes were not labeled. The percentage of silver grains over the Herring bodies increased with time. In rats deprived of water after fucose-³H injection there was a great increase in the release of labeled glycoproteins from the neurohypophysis. These results indicate that the glycoproteins synthesized by the secretory neurons of the hypothalamic nuclei are secreted in the neurohypophysis.     

Lipid binding-induced conformational change in human apolipoprotein E. Evidence for two lipid-bound states on spherical particles

Apolipoprotein (apo) E contains two structural domains, a 22-kDa (amino acids 1-191) N-terminal domain and a 10-kDa (amino acids 223-299) C-terminal domain. To better understand apoE-lipid interactions on lipoprotein surfaces, we determined the thermodynamic parameters for binding of apoE4 and its 22- and 10-kDa fragments to triolein-egg phosphatidylcholine emulsions using a centrifugation assay and titration calorimetry. In both large (120 nm) and small (35 nm) emulsion particles, the binding affinities decreased in the order 10-kDa fragment approximately 34-kDa intact apoE4 > 22-kDa fragment, whereas the maximal binding capacity of intact apoE4 was much larger than those of the 22- and 10-kDa fragments. These results suggest that at maximal binding, the binding behavior of intact apoE4 is different from that of each fragment and that the N-terminal domain of intact apoE4 does not contact lipid. Isothermal titration calorimetry measurements showed that apoE binding to emulsions was an exothermic process. Binding to large particles is enthalpically driven, and binding to small particles is entropically driven. At a low surface concentration of protein, the binding enthalpy of intact apoE4 (-69 kcal/mol) was approximately equal to the sum of the enthalpies for the 22- and 10-kDa fragments, indicating that both the 22- and 10-kDa fragments interact with lipids. In a saturated condition, however, the binding enthalpy of intact apoE4 (-39 kcal/mol) was less exothermic and rather similar to that of each fragment, supporting the hypothesis that only the C-terminal domain of intact apoE4 binds to lipid. We conclude that the N-terminal four-helix bundle can adopt either open or closed conformations, depending upon the surface concentration of emulsion-bound apoE.     

Unstable nuclear DNA in hypoglossal neurons of adult mice

To demonstrate the existence of unstable or metabolic DNA in normal mammalian neurons and to study the effect of peripheral nerve injury on this metabolic DNA, adult mice were given repeated injections of high doses of 3H-thymidine (3H-T) on the day before injury to the left hypoglossal nerve. The animals were killed at intervals up to 33 days after the injections of 3H-T. Analyses of grain counts showed a low but significant elevation in the number of radioautographic grains per unit area of hypoglossal neuronal nuclei above background levels for up to 5 days after 3H-T injection. Digestion of the tissue with DNase lowered the nuclear grain counts to background levels, confirming that the DNA was indeed labelled. Although there was a loss of labelled material from the neuronal nuclei with time, there was no difference between injured and uninjured neurons at any of the intervals tested after injection of 3H-T.     

The glycopeptide domain of the rat vasopressin precursor

The vasopressin precursor is composed of 3 domains, namely vasopressin, MSEL-neurophysin and a glycopeptide. Processing occurs during axonal transport from hypothalamus to neurohypophysis from which the 3 fragments can be isolated. The glycopeptide fragment of the rat vasopressin precursor has been purified and sequenced. Despite the fact that rat MSEL-neurophysin is shortened (93 residues instead of 95 for other mammals), rat glycopeptide has 39 residues, as do the other mammalian glycopeptides, suggesting a similar processing. Fifteen substitutions are however observed when compared to ox glycopeptide. The C-terminal part of MSEL-neurophysin (residues 77-93) and the glycopeptide are encoded by the same exon and the homologies when compared with their bovine counterparts are 58% and 62% respectively. In contrast, the central part of rat MSEL-neurophysin (residues 10-76), which is encoded by a separate exon, displays 96% of homology; vasopressin and the N-terminal part of MSEL-neurophysin (residues 1-9), encoded by a third exon, are nearly invariant.     

Phosphopeptides derived from hen egg yolk phosvitin: effect of molecular size on the calcium-binding properties.

Two different phosphopeptide (PPP) fragments derived from partially dephosphorylated hen egg yolk phosvitin were prepared by tryptic digestion, and their Ca2+ binding property compared with that of commercial casein phosphopeptides (CPP). The smaller fragment of less than 1 kDa and O-phospho-1-serine did not bind Ca2+ to any significant extend, while PPP of 1-3 kDa showed a higher ability than CPP to render soluble calcium. The results show that not only the phosphoserine residues are critical for Ca2+ binding, but also the molecular size of the phosphopeptides.     

Human complement component C4. Structural studies on the fragments derived from C4b by cleavage with C3b inactivator.

1. One of the activation products of C4, C4b, was prepared, and the reactive thiol group on the alpha'-chain was radioactively labelled with iodo[2-14C]acetic acid. The alpha'-chain was isolated and the N-terminal amino acid sequence of the first 13 residues was determined. 2. C4b was cleaved by C3bINA in the presence of C4b-binding protein and C4d and C4c isolated. The radioactive label and therefore the reactive thiol group were located to C4d. 3. C4c was reduced and alkylated and the two alpha'-chain fragments of C4c were separated. 3. The molecular weights, amino acid analyses and carbohydrate content of the three alpha'-chain fragments were determined. C4d has a mol.wt. of 44500 and a carbohydrate content of 6%. The two alpha'-chain fragments of C4c have mol.wts. of 25000 (alpha 3) and 12000 (alpha 4) and carbohydrate contents of 10 and 22% respectively. 4. The N-terminal amino acid sequences of C4d, the alpha 3 and the alpha 4 fragments were determined for 18, 24 and 11 residues respectively and, by comparison with the N-terminal sequence of the C4b alpha'-chain, the 25000-mol.wt. fragment (alpha 3) was shown to be derived from the N-terminal part of the alpha'-chain. 5. C-Terminal analyses were done on the alpha'-chain and its three fragments. Arginine was found to be the C-terminal residue of C4d and of the alpha 3 fragment. The C-terminal residue of the alpha'-chain and of the alpha 4 fragment could not be identified. The order of the three fragments of the alpha'-chain is therefore: alpha 3(25000)--C4d(44500)--alpha 4(12000). The specificity of C3bINA is for an Arg--Xaa peptide bond.     

Synthesis and in vitro bioactivity of C-terminal deleted analogs of human growth hormone-releasing factor

A series of C-terminal deleted analogs of human growth hormone-releasing factor (hGRF) with either an amidated or a free carboxylic acid C-terminus were synthesized by solid phase methodology. Their capacity to release growth hormone was tested on rat anterior pituitary cells in monolayer culture. A gradual decrease of bioactivity down to 23% relative to hGRF was noted when the C-terminal amino acids were deleted to hGRF (1-34)OH. Further deletions, however, did not decrease the bioactivity because the potencies of the fragments, hGRF(1-31)NH2, (1-30)NH2 and (1-29)NH2 remained at about 50% of that of hGRF. Continual deletion of residues to hGRF(1-23)NH2, (1-22)NH2 and (1-21)NH2 still yielded bioactive fragments with full intrinsic activity despite very low potency. Only with the deletion down to hGRF(1-19)NH2 did the bioactivity completely disappear. Thus, together with the data published in a previous paper (1), the minimal biologically active core of hGRF with full intrinsic activity comprises the fragment (3-21).     

Synthesis of membrane glycoproteins in rat small-intestinal villus cells. Redistribution of l-[1,5,6-3H]-fucose-labelled membrane glycoproteins among Golgi, lateral basal and microvillus membranes in vivo

The biogenesis of plasmalemma glycoproteins of rat small-intestinal villus cells was studied by following the incorporation of l-[1,5,6-(3)H]fucose, given intraperitoneally with and without chase, into Golgi, lateral basal and microvillus membranes. Each membrane fraction showed distinct kinetics of incorporation of labelled fucose and was differently affected by the chase, which produced a much greater decrease in incorporation of label into Golgi and microvillus than into lateral basal membranes. The kinetic data suggest a redistribution of newly synthesized glycoproteins from the site of fucosylation, the Golgi complex, directly into both lateral basal and microvillus membranes. The observed biphasic pattern of label incorporation into the microvillus membrane fraction may be evidence for a second indirect route of incorporation. The selective effect of the chase suggests the presence of two different pools of radioactive fucose in the Golgi complex that differ in (1) their accessibility to dilution with non-radioactive fucose, and (2) their utilization for the biosynthesis of membrane glycoproteins subsequently destined for either the microvillus or the lateral basal parts of the plasmalemma. The radioactively labelled glycoproteins of the different membrane fractions were separated by sodium dodecyl sulphate/polyacrylamide-slab-gel electrophoresis and identified by fluorography. The patterns of labelled glycoproteins in Golgi and lateral basal membranes were identical at all times. At least 14 bands could be identified shortly after radioactive-fucose injection. Most seemed to disappear at later times, although one of them, which was never observed in microvillus membranes, increased in relative intensity. All but two of the labelled glycoproteins present in the microvillus membrane corresponded to those observed in Golgi and lateral basal membranes shortly after fucose injection. The patterns of labelled glycoproteins in all membrane fractions were little affected by the chase. These data support a flow concept for the insertion of most surface-membrane glycoproteins of the intestinal villus cells.     

Labelling of amoeboid microglial cells in rats of various ages following an intravenous injection of horseradish peroxidase

The macrophagic amoeboid microglial cells in the corpus callosum of postnatal rats were labelled following an intravenous injection of horseradish peroxidase (HRP). The earliest time when these cells were labelled was 3 h after the injection of HRP in postnatal (1-10 days) rats. Similar cells around the mesencephalic aqueduct and the fourth ventricle were also labelled. These cells, however, were weakly labelled in developing (11-20 days) and unlabelled in weaning (21-30 days) rats. The results suggest that in the postnatal rats, the HRP passed through the endothelial lining of the blood vessels and was then ingested by the amoeboid microglial cells. In the developing and older rats, the wall of blood vessels had developed fully thereby preventing the free passage of HRP into the brain tissues.     

Synthesis of membrane glycoproteins in rat small-intestinal villus cells. Effect of colchicine on the redistribution of L-[1,5,6-3H]fucose-labelled membrane glycoproteins among Golgi, lateral basal and microvillus membranes.

To define the role of cytoplasmic microtubules in the biogenesis of plasmalemma glycoproteins of rat small-intestinal villus cells, we studied the effect of colchicine on the incorporation of L-[1,5,6-3H]fucose into Golgi, lateral basal and microvillus membranes. Colchicine was administered intraperitoneally before or after injection of radioactive fucose. The incorporation of radioactivity into Golgi membranes was little affected by colchicine, which did not prevent the redistribution of most of the labelled glycoproteins from the Golgi complex into other parts of the villus cell. The incorporation of labelled glycoproteins into the microvillus membrane was greatly inhibited by colchicine given 2 h or 10 min before the radioactive fucose: all labelled glycoproteins present in this membrane were equally affected. In contrast, the administration of colchicine considerably increased the incorporation of radioactivity into the lateral basal part of the plasmalemma, and prevented the disappearance of most of the labelled glycoproteins from this membrane at late times after fucose injection. These results suggest that cytoplasmic microtubular structures are important for the polarization of the intestinal villus cell and the biogenesis of the microvillus membrane, although playing little or no role in the movement of membrane components from the Golgi complex to the lateral basal part of the plasmalemma.