Neutralization of the infectivity of sporozoites of Plasmodium knowlesi by antibodies to a synthetic peptide.

Antibodies against a synthetic peptide representing the repetitive epitope of the circumsporozoite protein (CS) of Plasmodium knowlesi have properties similar to those of antibodies against the native protein. Either antibody reacts with the synthetic peptide, cross-links the CS protein on the membrane of the parasite giving the CSP reaction, and neutralizes the infectivity of sporozoites. The synthetic peptide and sporozoite extracts were equally effective when used in an immunoradiometric assay as antigens to detect antibodies to CS proteins. It is likely that the corresponding synthetic repeats from the human malaria parasites could be used to measure levels of anti-sporozoite antibodies in endemic areas, or to evaluate the humoral response to anti-sporozoite vaccines. The authors are grateful to Dr. Robert Gwadz, NIH, for supplying Anopheles mosquitoes and P. knowlesi sporozoites used in this study.

A synthetic pentapeptide with biological activity characteristic of the thymic hormone thymopoietin.

The pentapeptide arginyl-lysyl-aspartyl-valyl-tyrosine, corresponding to amino acid residues 32--36 in thymopoietin, was synthesized. In vitro, this pentapeptide induced the differentiation of murine prothymocytes to thymocytes and inhibited differentiative induction of cells of the B lineage. This combination of actions is presently unique to the parent molecule thymopoietin. In vivo, the pentapeptide reduced the high numbers of autologous rosette-forming cells normally present in the spleens of athymic mice; this also is a property of thymopoietin. These results suggest that this readily synthesized pentapeptide corresponds to an active site of thymopoietin and might serve as a therapeutic substitute for thymopoietin.

Structural characterization and localization of corticotropin-releasing factor in testis.

To sequence and thereby definitively characterize corticotropin-releasing factor (CRF)-like material from a representative peripheral tissue, CRF was obtained from 76 ovine testes. The novel extraction procedure involved use of an immunoaffinity column to which a high-affinity CRF monoclonal antibody was attached as well as fast protein liquid chromatography. The complete sequence was elucidated by gas-phase sequencing, carboxyamidopeptidase digestion and cyanogen bromide cleavage. Aside from microheterogeneity at position 39, all the other amino acids were identical to ovine hypothalamic CRF. Additionally, in immunohistochemical studies in the rat, CRF was localized to the Leydig cell. These findings along with related observations by ourselves and others are compatible with the hypothesis that CRF plays a significant local role, possibly by paracrine or autocrine mechanisms.

A seasonal variation in arginine vasotocin immunoactivity in rat pineal glands.

Groups of male and female laboratory rats, 28-30 days of age, were killed each week from July 1980 to September 1981. Pineal glands were collected, pooled, and extracted. Arginine vasotocin (AVT) activity in the extracts was measured by RIA. For most of the calendar year, pineal AVT immunoactivity ranged between 1.8-7.7 pg/gland. The average (+/- SE) basal AVT activity level was 4.1 +/- 0.3 pg/gland (n = 48). Both years in early August, pineal AVT activity increased several hundred fold. Values of 1720 and 1170 pg/gland were measured in mid-August of 2 successive years. The signal for this dramatic yearly rhythm, and its physiological consequences, are as yet unknown.

Biosynthesis of neurophysin proteins in the dog and their isolation.

Neurophysin (Np) is generally found in close association with vasopressin and oxytocin in the hypothalamo-neurohypophyseal complex. Dog neurophysin I and II have been isolated from fresh and frozen posterior pituitaries. The proteins were characterized on the basis of disc electrophoresis, immunological properties, amino acid composition and partial sequence determination. The amino terminal sequence of dog Np I is Ala-Ala-Leu-Asp-Leu-Asp-Val-Arg-Gln-Cys-Leu-Pro-Cys-Gly-Pro-Gly-Gly-Gln-Gly-while that of dog Np-II is Ala-Met-Ser-Asp-Leu-Glu-Leu. The dog Np I appears to be metabolically less stable than Np II. Isotope experiments with [35S]cystine or 3H-labeled amino acids using a design of "in vitro pulse and in vitro chase" as well as "in vivo pulse and in vivo chase," added further confirmation of the capability of the hypothalamic neurosecretory cells to synthesize concomitantly precursors of Np and vasopressin. The radioactively labeled precursors were converted to Np-like protein and vasopressin, both of which were isolated.

Corticotrophin-releasing factor in extra-hypothalamic brain of the mouse: demonstration by immunoassay and immunoneutralization of bioassayable activity.

Corticotrophin-releasing factor (CRF) bioactivity has been described in the extra-hypothalamic brain, but its relationship to hypothalamic CRF has remained questionable. Of the seven regions of the mouse brain examined, highest concentrations of CRF-like immunoreactivity (CRF-LI) and bioassayable CRF activity were present in the median eminence and hypothalamus. However, substantial CRF-LI and bioassayable CRF activity were also seen in brain extracts from the amygdala, thalamus, frontal cortex, pons medulla and cerebellum. Bioactivity was largely neutralized by prior incubation with heat-inactivated antiserum to ovine CRF. These findings, in conjunction with previous immunocytochemical evidence, strongly suggest that a substance closely resembling hypothalamic CRF is present in the extrahypothalamic brain of the mouse.

The effect of neuropeptide Y (NPY) on stimulation-evoked release of [3H]norepinephrine (NE) from rat hypothalamic and cerebral cortical slices.

The effects of neuropeptide Y (NPY) on stimulation-evoked release of [3H]norepinephrine ([3H]NE) in rat hypothalamic and cerebral cortical slices were investigated. NPY inhibits the stimulation-evoked release of [3H]NE from hypothalamic, but not from cerebral cortical slices. NPY potentiates the inhibition of [3H]NE release by the alpha 2-agonist UK 14,304 in the hypothalamic slices. The blockade of alpha 2-adrenoceptors by RX 781094 diminishes the inhibitory effects of NPY. These results suggest that in the hypothalamic slices the action of NPY might be in part mediated by the alpha 2-adrenoceptors.

Human salivary acidic proline-rich protein polymorphisms and biosynthesis studied by high-performance liquid chromatography.

Human salivary acidic proline-rich proteins (PRPs) constitute a significant fraction of the total salivary protein and possess important biological activities. Different genetic and post-translationally processed forms of the PRPs exhibit significant quantitative variations in several of these activities, especially the modulation of salivary calcium phosphate chemistry and oral bacterial adhesion. To quantify and understand these differences, we have developed a high-performance liquid chromatography (HPLC) method to identify and measure individual PRPs in saliva. The data obtained permit the identification of PRP polymorphisms and phenotypes, the determination of the relative amounts of PRPs derived from the two loci, PRH1 and PRH2, and the measurement of the extent of post-translational cleavage of the primary polypeptide products. Substantial inter-gland and inter-individual variations were found in relative amounts of PRPs derived from the two loci (at least two-fold), and in post-translational cleavage (greater than two-fold), both of which are likely to be biologically significant. Also in this study, the presence of what appear to be minor amounts of numerous variant PRPs in glandular secretions was observed, and two uncommon PRP polymorphisms were identified in the 127 subjects studied.

Complete covalent structure of a proline-rich phosphoprotein, PRP-2, an inhibitor of calcium phosphate crystal growth from human parotid saliva.

Human salivary secretions contain many proteins in which proline forms an unusually large fraction of the amino-acid residues present, typically from 20% to over 40%. These proteins are also unusually rich in glycine and glutamine, generally account for over half the total protein in saliva, and include acidic, basic and glycosylated molecules. The functions of most of these are not clearly defined. One group, however, the acidic proline-rich phosphoproteins (PRP), have been shown to be potent inhibitors of secondary precipitation (crystal growth) of calcium phosphate salts. Acting together with a salivary protein inhibitor of primary precipitation of calcium phosphates, statherin, the PRP stabilize saliva which is supersaturated with respect to the calcium phosphate salts which form dental enamel. These inhibitory activities act to provide a protective, reparative, but stable environment for dental enamel, which is important for maintaining the health of the teeth. The PRP are a complex group of phosphoproteins which include four major and at least eight minor members. The primary structures of three of the major proteins have been determined. These are PRP-1, also designated Protein-C, PRP-3, also designated Protein-A (17), and PRP-4. The designations PRP-1,-2,-3 and -4 will be used here. The purpose of this paper is to report the complete primary structure of PRP-2 as a further step towards establishing the structural basis of the biological activity of the PRP, and clarifying the genetic and biosynthetic relationships of these closely related proteins.

Primary structure of the active tryptic fragments of human and monkey salivary anionic proline-rich proteins.

The primary structures of the four human salivary anionic proline-rich proteins and an analogous protein from the saliva of a monkey (Macaca arctoides) have been further investigated. These proteins possess the unusual property of inhibiting crystal growth of calcium phosphate salts, and it has been proposed that they play an important role in the mouth, by preventing precipitation of calcium phosphate salts from the supersaturated saliva. The tryptic fragments responsible for this activity have been isolated from all five proteins and their complete amino acid sequences determined and compared. These active fragments have been unequivocally identified as the amino-terminal segment in all five proteins. The structures of the four human fragments are identical except for the presence of Asn at residue 4 in PRP-1 and -3 instead of Asp found in PRP-2 and -4. Comparison of the 30 residue human fragments with the monkey fragment shows 18 residues to be identical in these peptides, providing that residue 1 of the monkey fragment is aligned with residue 3 of the human proteins. Theses studies constitute the next step in determining the mechanism of action of these unusual proteins, and in determining their minimum chain length required for inhibitory activity.

The amino acid sequence of thymopoietin II.

The amino acid sequence of bovine thymopoietin II is presented. This T cell differentiating hormone of the thymus is a single 49 amino acid polypeptide chain of 5562 daltons. There is microheterogeneity at the C terminus with approximately two thirds of the molecules lacking the C terminal arginine found on the remaining molecules. Determination of the primary structure of thymopoietin II was facilitated by a long automated sequenator run on thymopoietin II coupled to 2-isothiocyanonaphthalene-4,8-disulfonic acid (NITC), tryptic cleavage of maleated thymopoietin II to yield the overlapping C terminal peptide, and efficient manual sequencing of this peptide using benzene extractions to minimize extractive losses of peptide.

Thymopoietin and myasthenia gravis: neostigmine-responsive neuromuscular block produced in mice by a synthetic peptide fragment of thymopoietin.

Thymopoietin is the thymic hormone responsible for inducing the differentiation of thymocytes. A synthetic peptide corresponding to positions 29-41 of bovine thymopoietin II, which can reproduce this function of the parent molecule in vitro, also caused neostigmine-responsive neuromuscular block in mice similar to that which thymopoietin itself produces, and which resembles the neuromuscular block of myasthenia gravis. This 13 aminoacid sequence must therefore contain the key residues in the thymopoietin molecule responsible for both T-cell differentiation and neuromuscular effects. These data provide further support for the view that myasthenia gravis is a consequence of thymic disease associated with systemic release of thymopoietin, with ensuing neuromuscular block. This evidence supports the empirically proven treatment of myasthenia gravis by early total thymectomy.

Complete covalent structure of statherin, a tyrosine-rich acidic peptide which inhibits calcium phosphate precipitation from human parotid saliva.

The complete amino acid sequence of human salivary statherin, a peptide which strongly inhibits precipitation from supersaturated calcium phosphate solutions, and therefore stabilizes supersaturated saliva, has been determined. The NH2-terminal half of this Mr=5380 (43 amino acids) polypeptide was determined by automated Edman degradations (liquid phase) on native statherin. The peptide was digested separately with trypsin, chymotrypsin, and Staphylococcus aureus protease, and the resulting peptides were purified by gel filtration. Manual Edman degradations on purified peptide fragments yielded peptides that completed the amino acid sequence through the penultimate COOH-terminal residue. These analyses, together with carboxypeptidase digestion of native statherin and of peptide fragments of statherin, established the complete sequence of the molecule. The 2 serine residues (positions 2 and 3) in statherin were identified as phosphoserine. The amino acid sequence of human salivary statherin is striking in a number of ways. The NH2-terminal one-third is highly polar and includes three polar dipeptides: H2PO3-Ser-Ser-H2PO3-Arg-Arg-, and Glu-Glu-. The COOH-terminal two-thirds of the molecule is hydrophobic, containing several repeating dipeptides: four of -Gn-Pro-, three of -Tyr-Gln-, two of -Gly-Tyr-, two of-Gln-Tyr-, and two of the tetrapeptide sequence -Pro-Tyr-Gln-Pro-. Unusual cleavage sites in the statherin sequence obtained with chymotrypsin and S. aureus protease were also noted.

Delineation of a segment of adsorbed salivary acidic proline-rich proteins which promotes adhesion of Streptococcus gordonii to apatitic surfaces.

Cells of several strains of Streptococcus gordonii attached in much higher numbers to experimental pellicles formed from samples of submandibular or parotid saliva on hydroxyapatite (HA) beads than to buffer controls. The nature of the salivary components responsible were investigated by preparing experimental pellicles from chromatographic fractions of submandibular saliva obtained from Trisacryl GF 2000M columns. Adhesion of S. gordonii Blackburn was promoted by two groups of fractions. The adhesion-promoting activity in the first group of fractions was associated with the family of acidic proline-rich proteins (PRPs), while that of the second group is as yet unidentified. Experimental pellicles prepared by treating HA with 2 micrograms of pure 150-amino-acid-residue PRPs (PRP-1, PRP-2, and PIF-s) promoted adhesion of S. gordonii Blackburn cells to an extent comparable to that obtained with unfractionated saliva. However, pellicles prepared from a 106-residue PRP (PRP-3) were significantly less effective, and those prepared from the amino-terminal tryptic peptide (residues 1 to 30) of the PRP and the salivary phosphoprotein statherin were completely ineffective in promoting adhesion. Although adhesion of several strains of S. gordonii was promoted by adsorbed PRP-1, the adhesion of several strains of Streptococcus sanguis or Streptococcus oralis was either not affected or only weakly enhanced by this protein. S. gordonii cells bound avidly to PRPs adsorbed onto HA beads, but the streptococci did not appear to bind PRPs in solution, since concentrations of PRP as high as 200 micrograms/ml did not inhibit binding of bacterial cells to pellicles prepared from pure PRP. S. gordonii cells also attached well to PRP or a synthetic decapeptide representing residues 142 to 150 of the PRP when the peptide was linked to agarose beads. Studies with a series of synthetic decapeptides indicated that the minimal segment of PRP which promoted high levels of S. gordonii adhesion was the carboxy-terminal dipeptide Pro-Gln (residues 149 and 150).

Isolation and complete amino acid sequence of human thymopoietin and splenin.

Human thymopoietin and splenin were isolated from human thymus and spleen, respectively, by monitoring tissue fractionation with a bovine thymopoietin RIA cross-reactive with human thymopoietin and splenin. Bovine thymopoietin and splenin are 49-amino acid polypeptides that differ by only 2 amino acids at positions 34 and 43; the change at position 34 in the active-site region changes the receptor specificities and biological activities. The complete amino acid sequences of purified human thymopoietin and splenin were determined and shown to be 48-amino acid polypeptides differing at four positions. Ten amino acids, constant within each species for thymopoietin and splenin, differ between the human and bovine polypeptides. The pentapeptide active site of thymopoietin (residues 32-36) is constant between the human and bovine thymopoietins, but position 34 in the active site of splenin has changed from glutamic acid in bovine splenin to alanine in human splenin, accounting for the biological activity of the human but not the bovine splenin on the human T-cell line MOLT-4.