Cardioactive Protein-Hormonal Complexes of Brain and Heart

New cardioactive protein–hormone complexes (PHC) are identified in magnocellular nuclei of hypothalamus. It was proved that they are specific for nervous tissues and are involved in the regulation of metabolic processes of brain and visceral organs, including the heart. PHC dissociate into high-molecular forms which are new specific glycoproteins and the low-molecular cardioactive neurohormones. Results of our own studies on the functional activities of PHC as well as cardioactive peptides in the precardiac and auricular regions of the heart with respect to the parameters of haemostasis system are reviewed.     

The effects of small cardioactive peptide B on the isolated heart and gill of Aplysia californica

Effects of small cardioactive peptide B on the physiology of the isolated heart and gill preparations from the mollusc Aplysia californica were examined. In addition, the effects of small cardioactive peptide B and FMRFamide (Phe-Met-Arg-Phe-NH2) on adenylate cyclase activity were compared in particulate fractions of heart and gill tissues, respectively. Small cardioactive peptide B was found to exert dose-dependent, reversible changes in cardiac activity when perfused through the isolated heart. The EC50 values effecting changes in heart rate and force of contraction were 3 X 10(-11) and 3 X 10(-10) M, respectively; minimum concentrations found to effect changes in heart rate and force of contraction were normally 10(-15) and 10(-12) M, respectively. However, some winter hearts demonstrated threshold sensitivity to small cardioactive peptide B at concentrations as low as 10(-17) M. When perfused through the isolated gill, small cardioactive peptide B was found to suppress the gill withdrawal response amplitude with a threshold concentration of 10(-14) M and an EC50 value of 3 X 10(-11) M. Suppression of the gill withdrawal response amplitude by small cardioactive peptide B was found to be dose dependent and reversible up to a concentration of 10(-9) M. At higher concentrations, the suppression tended to persist irreversibly. Small cardioactive peptide B stimulated adenylate cyclase activity in particulate fractions of both heart and gill tissues with an EC50 of 0.1 and 1.0 microM, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)     

Cardioactive neuropeptides in gastropods

At least five neuropeptides that are active on an isolated snail heart can be recovered from extracts of gastropod nervous tissue. These peptides have been divided into three classes. The class of the lowest molecular weight, termed the small cardioactive peptides (SCPs), is made up of two peptides. SCPs have been found in all gastropods studied and appear to be involved in the control of the gut. They have been localized by microdissection and bioassay to several identified central neurons that send their axons out to innervate the gut. These neurons act centrally to enhance the motor output of the ganglia responsible for the control of feeding, and peripherally to modulate gut activity. In one pair of these neurons, the classical transmitter acetylcholine coexists with an SCP. The next larger peptide class (medium cardioactive peptide), found only in Aplysia, shares both its mode of cardiac activity and tissue distribution with the SCPs. As yet, there is no evidence that either of these peptide classes acts as a physiological modulator of cardiac activity. The class of the highest molecular weight (large cardioactive peptide [LCPs]) is made up of two peptides and is found only in Helix. The LCPs are circulating neurohormones involved in the regulation of heart, gut, and neuromuscular activity. Their primary release site is a neurohemal region in the auricle. The significance of these findings is discussed in light of recent advances in the study of mammalian neuropeptides.     

Precardiac cell migration: fibronectin localization at mesoderm-endoderm interface during directional movement

The pathway of directional movement of chick precardiac mesoderm cells was studied by indirect immunofluorescence and by scanning electron microscopy. Directional movement of the precardiac cells begins at stage 6 from the lateral sides of the embryo at the level of Hensen's node. The cells move anteriorly in an arc to the embryo's midline. By stage 8 the cells arrive at the lateral sides of the anterior intestinal portal and movement ceases. The interval of this directional movement is approximately 10 hr. During migration the precardiac cells are in close association with the underlying endoderm. As migration proceeds, the cells encounter increasing amounts of fibrils in the substratum at the mesoderm-endoderm interface. Concomitant with increasing fibril formation there is an increase in fibronectin (FN) in the heart-forming region. During stage 5 FN first appears in the lateral heart-forming regions and increases in amount during the period of cell migration. By stage 7 a concentration difference of FN is apparent in the lateral regions with more FN cephalad and decreasing amounts caudad. At stages 7 and 8 large amounts of extracellular FN-associated fibrils are observed at the lateral sides of the anterior intestinal portal where the cells stop moving. The precardiac cells moving into this region are oriented perpendicular to the anterior intestinal portal and in close association with these fibrils. There is no evidence that the fibrillar meshwork forming the substratum of the precardiac mesoderm cells is physically oriented as a guide for directional movement. The correlations between FN distribution at the mesoderm-endoderm interface and directional cell movement suggest that the precardiac cells may migrate by haptotaxis, i.e., by moving along the substratum toward areas of greater adhesiveness.     

Amide proton exchange rates in cardioactive sea anemone polypeptides

Amide hydrogen exchange rates have been measured using high-resolution 1H nuclear magnetic resonance (NMR) spectroscopy at 300 MHz for three homologous cardioactive polypeptides, anthopleurin-A from Anthopleura xanthogrammica and Anemonia sulcata toxins I and II. There are approximately 15 slowly exchanging hydrogens in each polypeptide, but the slowest exchange rates are found in ATX II, with ATX I and AP-A having rates similar to one another. The exchange rates correlate with the thermal stability of these molecules, but not with the potency and species specificity of their biological activities. The data for AP-A are interpreted in terms of a recent structural model for this polypeptide.     

Isolation and structure of corazonin, a cardioactive peptide from the American cockroach

Corazonin, a new cardioaccelerating peptide, has been isolated from the corpora cardiaca of the American cockroach, Periplaneta americana, and its structure determined to be Glp-Thr-Phe-Gln-Tyr-Ser-Arg-Gly-Trp-Thr-Asn-amide. The peptide stimulated heart beat at concentrations as low as 0.2 nM, which makes it the most potent insect cardioactive neuropeptide.     

The effects of retinoic acid on heart formation in the early chick embryo.

The vitamin A derivative retinoic acid has previously been shown to have teratogenic effects on heart development in mammalian embryos. The craniomedial migration of the precardiac mesoderm during the early stages of heart formation is thought to depend on a gradient of extracellular fibronectin associated with the underlying endoderm. Here, the effects of retinoic acid on migration of the precardiac mesoderm have been investigated in the early chick embryo. When applied to the whole embryo in culture, the retinoid inhibits the craniomedial migration of the precardiac mesoderm resulting in a heart tube that is stunted cranially, while normal or enlarged caudally. Similarly, a local application of retinoic acid to the heart-forming area disrupts the formation of the cardiogenic crescent and the subsequent development of a single mid-line heart tube. This effect is analogous to removing a segment of endoderm and mesoderm across the heart-forming area and results in various degrees of cardia bifida. At higher concentrations of retinoic acid and earlier developmental stages, two completely separate hearts are produced, while at lower concentrations and later stages there are partial bifurcations. The controls, in which the identical operation is carried out except that dimethyl sulphoxide (DMSO) is used instead of the retinoid, are almost all normal. We propose that one of the teratogenic effects of retinoic acid on the heart is to disrupt the interaction between precardiac cells and the extracellular matrix thus inhibiting their directed migration on the endodermal substratum.     

Relation between the Yersinia phage and bacteriophages isolated from the environment

The bacteriophage designated RD2 has been isolated from the sewage in Rostov-on-Don city and studied. The morphology of bacteriophage particles and the biological properties of the bacteriophage make it related to the plague bacteriophage isolated by D'Errel. The molecular masses of the compared bacteriophages are almost identical being 26.4 +/- 0.4 Md for RD2 and 24.7 +/- 0.2 Md for D'Errel bacteriophage. The DNAs of the bacteriophages share 80% of homology and possess 15 nonhomologous regions scattered along the genomes. The phages are serologically related. The DNAs of both bacteriophages give the similar pattern of hydrolysis by restriction endonuclease EcoRV, but have the different sensitivity to many other restriction endonucleases. The protein specter of bacteriophage RD2 contains 18 polypeptides (11 minor ones), while the one of D'Errel bacteriophage contains 7 polypeptides similar in molecular mass with the polypeptides of RD2. The bacteriophage RD2 cannot be considered one of the plague causative agents of bacteriophages since the region where it has been isolated has a long epidemiological and epizootical record of absence of plague.     

Effects of pH and temperature on cardioactive polypeptides from sea anemones: a 1H-NMR study

The effects of pH and temperature on the 300-MHz ¹H-nmr spectra of three cardioactive polypeptides from sea anemones, anthopleurin-A from Anthopleura xanthogrammica (AP-A) and Anemonia sulcata toxins I and II (ATX I and II), are described. AP-A and ATX II exhibit major spectral heterogeneity. Evidence from the pH and temperature studies and from high performance liquid chromatography indicates that this heterogeneity is conformational rather than chemical in origin. By contrast, purified isotoxins of ATX I show no evidence of conformational heterogeneity. The pKa values of most of the ionizable groups in these polypeptides are not strongly perturbed by interactions in the tertiary structure, with the exception of one of the Asp carboxylates, which has a pKa of ? 2 in AP-A and ATX II and 3.0 in ATX I. Protonation of this carboxylate, suggested to be Asp-9, leads to a conformational change in all three molecules. All three polypeptides are thermally stable, showing some conformational changes but not major unfolding at elevated temperatures.     

Two major antigens of heart sarcolemma are Ca2(+)-binding glycoproteins that copurify with the dihydropyridine receptor.

Ca2+ binding has been studied in isolated heart sarcolemmal membranes using the 45Ca overlay technique. 45Ca bound to two sarcolemmal polypeptides of 125 kDa and 97 kDa in preparations from dog, rabbit, cow and pig. During fractionation on DEAE ion-exchange and wheat-germ lectin affinity columns, the two Ca2(+)-binding polypeptides copurified with the dihydropyridine receptor associated with the voltage gated Ca2+ channel. These polypeptides were the major proteins in the isolated fraction as judged by silver staining in SDS-PAGE. Antisera raised against purified dog heart, sarcolemma indicated that the 125 and 97 kDa polypeptides were highly antigenic components of this membrane. The antisera cross-reacted with similar polypeptides in cardiac sarcolemmal preparations from rabbit, cow and pig, but not sarcoplasmic reticulum membranes. Purified antibodies against the 125 kDa polypeptide did not cross-react with the 97 kDa polypeptide, while antibodies against the 97 kDa polypeptide did not cross-react with the 125 kDa polypeptide. Both the 125 kDa and 97 kDa polypeptides bound wheat-germ lectin, suggesting both were glycoproteins. It is unlikely that these Ca2+ binding glycoproteins represent subunits of the dihydropyridine receptor-Ca2+ channel in this membrane.     

Putative neurohemal areas in the peripheral nervous system of an insect,Gryllus bimaculatus,revealed by immunocytochemistry

The morphology and position of putative neurohemal areas in the peripheral nervous system (ventral nerve cord and retrocerebral complex) of the cricket Gryllus bimaculatus are described. By using antisera to the amines dopamine, histamine, octopamine, and serotonin, and the neuropeptides crustacean cardioactive peptide, FMRFamide, leucokinin 1, and proctolin, an extensive system of varicose fibers has been detected throughout the nerves of all neuromeres, except for nerve 2 of the prothoracic ganglion. Immunoreactive varicose fibers occur mainly in a superficial position at the neurilemma, indicating neurosecretory storage and release of neuroactive compounds. The varicose fibers are projections from central or peripheral neurons that may extend over more than one segment. The peripheral fiber varicosities show segment-specific arrangements for each of the substances investigated. Immunoreactivity to histamine and octopamine is mainly found in the nerves of abdominal segments, whereas serotonin immunoreactivity is concentrated in subesophageal and terminal ganglion nerves. Immunoreactivity to FMRFamide and crustacean cardioactive peptide is widespread throughout all segments. Structures immunoreactive to leucokinin 1 are present in abdominal nerves, and proctolin immunostaining is found in the terminal ganglion and thoracic nerves. Codistribution of peripheral varicose fiber plexuses is regularly seen for amines and peptides, whereas the colocalization of substances in neurons has not been detected for any of the neuroactive compounds investigated. The varicose fiber system is regarded as complementary to the classical neurohemal organs.     

A comparative study of the effect of abscisic acid and cAMP on protein synthesis in wheat caryopses under drought conditions

The effect of exogenous abscisic acid and cAMP on synthesis of soluble proteins in wheat caryopses in drought has been studied. Both compounds affected the formation of the polypeptides whose synthesis was stimulated by dehydration: they increased the incorporation of the label into polypeptides of 13, 15, and 26 kD and decreased the incorporation of the label into polypeptides of 14, 64, and 77 kD. Abscisic acid and cAMP increased the level of the incorporation of [¹⁴C]leucine into the low-molecular-weight polypeptides of 12, 17, and 19 kD whose synthesis was suppressed by drought. These data suggest that the cyclic adenylate signal system is probably involved in the effect of abscisic acid on protein synthesis in drought.     

The effect of 5-bromodeoxyuridine (BrdU) on cardiac muscle differentiation

Cultured cardiac muscle cells undergo cell division and form beating progeny. Incorporation of BrdU into the nuclei of daughter cells does not suppress their ability to beat and form cross-striated myofibrils. Fluorescence microscopy of clones derived from single beating cells fed with BrdU-treated medium for over 2 weeks reveal cytoplasmic fibrils stainable with fluorescein-labeled antimyosin. The effect of BrdU on the emergence of cardiac muscle phenotype was also investigated by utilizing cardiac myogenic precursor cells from precardiac mesoderm in early embryos (stage 4–stage 9). These studies show that the cardiac myogenic cells fall into the following categories with respect to their ability to express the differentiated phenotype in the presence of BrdU: (1) precardiac mesodermal cells that are inhibited; (2) precardiac mesodermal cells that are not inhibited; and (3) beating cardiac muscle cells that are not inhibited. The entry of precardiac cells from the first category to the second and to the third appears to be unsynchronized.     

Synthesis and Assembly of the Polypeptides of Photosystem I and II in Isolated Etiochloroplasts of Wheat

Synthesis and assembly of photosystems (PS) I and II polypeptides in etiochloroplasts isolated from greening wheat (Triticum aestivum L. cv Norin 61) seedlings were studied. The isolated etiochloroplasts synthesized PSI polypeptides of 66 and 15 kilodaltons, PSII polypeptides of 46 and 42 kilodaltons, and atrazine-binding 34 to 32 kilodalton polypeptide. Their assembly processes in the thylakoid membrane were studied by pulse-chase labeling with [³⁵S]methionine, mild solubilization of the thylakoid membrane with Triton X-100, sucrose density gradient centrifugation, and polyacrylamide gel electrophoresis. The newly synthesized polypeptides of 66, 46, 42, 34, and 32 kilodaltons were first integrated into the complexes of 7.5, 5.9, 7.5, 6.3, and 7.5 Svedberg units, respectively, in 20 minutes. After the chase with excess amount of methionine for 100 min, they were found in complexes of 9.5, 9.1, 9.1, 9.1, and 9.1 Svedberg units, respectively. In this condition, stained polypeptides of PSI and PSII were found in the complexes of 11.1 and 10.3 Svedberg units, respectively. These results indicated that newly synthesized PSI or PSII polypeptides are integrated into intermediate complexes, but not complete complexes in the isolated etiochloroplasts. The relationship between the processing of the atrazine-binding 32 kilodalton polypeptide and its assembly into the PSII complex is also discussed.     

Development of left/right handedness in the chick heart.

The chick heart tube develops from the fusion of the right and left areas of precardiac mesoderm and in almost all cases loops to the embryo's right-hand side. We have investigated whether any intrinsic difference exists in the right and left areas of precardiac mesoderm, that influences the direction of looping of the heart tube. Chick embryos incubated to stages 4,5 and 6 were cultured by the New method. Areas of precardiac mesoderm were exchanged between donor and host embryos of the same stage and different stages to form control, double-right and double-left sided embryos. Overall, double-right sided embryos formed many more left-hand loops than double-left sided embryos. At stages 4 and 5 a small percentage of double-right embryos formed left-hand loops (13%) whereas at stage 6 almost 50% of hearts had left-hand loops. Control embryos formed right-hand loops in 97% of cases. The stability of right-hand heart looping by double-left sided embryos, may be related to the process of 'conversion', whereas the direction of looping by double-right sided embryos has become randomised. There is some indication that an intrinsic change occurred in the precardiac mesoderm between stages 5 and 6 that later influenced the direction of looping of the heart tube. The direction of body turning is suggested to be linked to the direction of heart looping.     

Protein biosynthesis in a homologous, cell-free system in the presence of chick embryo RNA isolated from free and membrane-bound polyribosomes.

Protein synthesis has been studied in a cell-free system from chick embryo, in the presence of homologous RNA isolated from free and endoplasmic reticulum-bound polyribosomes. The two RNA fractions showed equal activities in total protein synthesis. However, while the RNA from bound polyribosomes mainly supported synthesis of high molecular weight, TCA-insoluble polypeptides, the RNA from free polyribosomes was more active in the synthesis of low molecular weight, TCA-soluble polypeptides. Optimal conditions for translation of the two RNA's under study were different when studied in a cell-free system with reduced content of endogenous matrix. Collagen synthesized in the system was identified by collagenase digestion. Collagen synthesis was demonstrated only in the presence of RNA from endoplasmic reticulum-bound polyribosomes, and represented 16-19% of total protein synthesis.     

Properties of thiamin-binding proteins from sesame seed 2S albumins

Three thiamin-binding proteins (TBPs) from sesame ( Sesamum indicum L.) seeds (STBP-I, -II and -III) were characterized. Binding of thiamin to the three STBPs was inhibited by pyrithiamin, which did not inhibit the binding of thiamin to TBPs from other plant seeds. STBP-I alone bound 2-northiamin and hydroxyethylthiamin. Isoelectric points (pIs) of STBP-I and -II both were 7.5. The pI of STBP-III was 6.5. STBPs did not have immunological homology with TBPs from rice seeds and buckwheat seeds. On the other hand, the amino acid compositions of the small and large polypeptides isolated from STBP-I, -II and -III resembled each other. Both the polypeptides contained large amounts of Glu (or Gln) and Arg. The small polypeptides contained more Ser than the large polypeptides. The N-terminal amino acid sequences (the first 29 residues) of the small polypeptides were identified. The N-terminal amino acid sequences of the three STBPs were the same. The small polypeptides had homology to castor bean 2S albumin small subunit. These results showed that STBPs were part of a plant protein superfamily and that STBPs differed from the TBPs of other plant seeds as to the binding to thiamin-related compounds and immunological properties, and further, that STBP-I, -II and -III differed in the affinity for thiamin-related compounds and pI, indicating that STBP-I, -II and -III are isomers.     

Analysis of Toxoplasma gondii proteins after Triton X-114 solubilization and hydrophobic chromatography

The distribution of the surface proteins of Toxoplasma gondii radiodinated were studied using the phase separation technique and ability of binding in the phenyl-Sepharose column. Eight polypeptides with Mr 22 to 180 distributed exclusively in the detergent rich-phase, while six polypeptides with mol. wt. 15,000 to 76,000 distributed exclusively in the detergent poor-phase. Two polypeptides with 15,000 and 70,000 distributed in both phase. All the polypeptides present in the detergent rich-phase binding in the phenyl-Sepharose column, and can be isolated in two peak according with their relative hydrophobicities. Two polypeptides hydrophobic with Mr 60 and 66 recognized by human serum were isolated by the association of the two technique. Our result showed that the surface proteins of T. gondii present different degrees of hydrophobicity and that the use of hydrophobic interaction chromatography after Triton X-114 extraction may be an important isolation method of membrane proteins.