Biomedical subjects
Publications and source records attributed to F Nyberg.
A new procedure using fast atom bombardment mass spectrometry for the determination of arginine residues in neuropeptides is described. The technique is based on the modification of the arginine side-group with 1,2-cyclohexanedione. This novel procedure may have a potential use in the study of neuropeptides and their processing and degrading enzymes.
Retinae from neonatal mice exposed to prolonged hyperoxia (100% oxygen), show marked vasoproliferation. Extracts of such retinae were chromatographed on a heparin-Sepharose column and the absorbed material subjected to HPLC fractionation. Two components, approx. 10 and 18 kDa, respectively, were found to have angiogenic activity, which was higher than in corresponding extracts from animals exposed to air. Both fractions and an additional 5 kDa component reacted with an antibody to basic fibroblast growth factor (bFGF) and showed higher levels in hyperoxia. The data suggest that hyperoxia activates angiogenic factors belonging to the heparin binding family.
This study describes the purification and characterization of an aminopeptidase from human cerebrospinal fluid capable of degrading delta-sleep-inducing-peptide (DSIP). The enzyme has an apparent molecular weight of approximately 80,000 dalton. It is sensitive towards amastatin, bestatin and EDTA and is optimally active at neutral pH. The recovered enzyme was also found to degrade other neuropeptides, e.g., the enkephalins.
Immunoreactive prolactin has been recovered from human cerebrospinal fluid (CSF) by hydrophobic interaction chromatography on Phenyl-Sepharose CL-4B and molecular sieving on Sephadex G-100. The hormone was obtained in a yield of about 1 microgram/liter CSF and was further analyzed by column electrophoresis on granular agarose at different pHs. All separation procedures were guided by radioimmunoassay. Upon chromatography on Sephadex G-100, the CSF prolactin coeluted with a pituitary reference preparation (molecular weight, 22,000 Da). A further relationship between the CSF prolactin and the pituitary hormone was demonstrated by the electrophoretic experiments. Thus, at alkaline pH, both preparations resolved into 3 or 4 active components, whereas only a single peak was observed in each preparation following runs performed at acidic pH. The results suggest that the prolactin activity present in the CSF specimens is identical with the hormone derived from the pituitary.
The distribution of substance P was determined in the rat spinal cord and brain after a focal traumatic injury to the thoracic region (T10-11) of the spinal cord. There was at 1 and 2 h after the injury a statistically significant increase of the substance P content not only in the injured segment but also in samples removed 5 mm proximal (T9) and distal (T12) to the lesion. At 5 h the substance P content of the injured segment of the cord was reduced by 30% compared with controls. However, there was a significant increase in the concentration of this peptide in segments located 5 mm cranial and caudal to the injury (65% and 22%, respectively). Interestingly, the whole brain content of substance P showed a statistically significant 22% increase from control values at 5 h after the injury. At 1 and 2 h after the spinal cord injury there was a significant decrease in whole brain substance P concentration by 25% and 65%, respectively. Pretreatment with p-chlorophenylalanine (a serotonin synthesis inhibitor) markedly reduced the endogenous content of substance P in whole brain of normal animals. In these animals, the spinal cord content of the peptide was elevated by 83-123% as compared to untreated control animals. Spinal cord trauma inflicted on p-chlorophenylalanine-treated animals did not affect the brain peptide level at all. However, a profound decrease was noted in all the spinal cord segments at 5 h as compared to the untreated traumatized group. The decrease in this peptide was more pronounced in the cranial and the injured segments as compared to the caudal one.(ABSTRACT TRUNCATED AT 250 WORDS)
Immunoreactive substance P was recovered from human brain (hypothalamus and substantia nigra) by acetic acid extraction, ion exchange chromatography (SP-Sephadex), molecular sieving (Sephadex G-50) and column electrophoresis in agarose suspension. The chemical nature of the active material was further studied with various biochemical techniques including agarose suspension electrophoresis, HPLC and different kinds of enzyme radioimmunoassays. By combining these techniques it was possible to confirm structure identity between the recovered active component and substance P previously isolated from bovine brain. Thus, the major activity reacting with the substance P antibodies was indistinguishable from the synthetic bovine analogue in all chromatographic systems including analytical electrophoresis at different pH:s and HPLC. Furthermore, digestion of the active material with post-proline cleaving enzyme and trypsin yielded fragments identical with those expected from the bovine peptide as confirmed by specific radioimmunoassays in conjunction with electrophoresis or HPLC. The result also indicates the usefulness of the present procedures for identifying peptides structures available only in minute amounts.
Pain experience and the amount of analgesics needed during labor were studied in 32 primiparous women who had received repeated treatment with acupuncture (AP) during the month prior to term and in 16 nontreated primiparous women. The women's psychological profiles were evaluated by a psychiatric interview at week 38 of pregnancy. Treatment with AP did not reduce the need for analgesics in labor. During labor, all women experienced successively rising pain irrespective of whether or not they had been treated with AP prior to labor or delivered under local anesthesia. Experience of pain was not reduced in subjective assessments in women treated with AP. There was a strong correlation between assessments of pain made during labor and 6 months after delivery. In the group that did not receive AP, cerebrospinal fluid dynorphin A was significantly lower in parturients who chose epidural anesthesia.
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This study describes three different endopeptidases in human spinal cord with high affinity for the dynorphins. The enzymes, with strict specificity towards paired basic residues, release Leu-enkephalin and its C-terminal extensions - Arg6 and -Arg6 -Arg7 from the substrate peptides. They differ with regard to molecular size, charge properties, and inhibitory profile. Two of the enzymes show optimal activity at neutral pH, whereas the third enzyme has its activity optimum at pH 5.6. They are all suggested to be of importance for the formation of Leu-enkephalin from prodynorphin.
Rats were given a continuous subcutaneous infusion at constant rate with either morphine, the opioid agonist/antagonist analgesic dezocine or saline. Tolerance to the antinociceptive effect of morphine or dezocine was complete on day 8, when cisterna magnum cerebrospinal fluid (CSF) was sampled under anesthesia. The activity of the enzymes cleaving dynorphin A (DCE) and substance P (SPE) was measured in the CSF. It was found that the animals treated with morphine had a 2- to 3-fold increase in both DCE and SPE activities. The animals treated with dezocine showed a similar increase in the activity of DCE, whereas SPE did not significantly change. These enzymes may therefore play a role in the development of tolerance to opioid analgesic drugs. The experiments show that chronic opioid treatment affects peptidergic mechanisms.
An endopeptidase that converts the opioid peptide dynorphin B (Tyr-Gly-Gly-Phe-Leu-Arg-aRg-Gln-Phe-Lys-Val-Val-Thr) to its bioactive fragment Leu-enkephalin-Arg6 was isolated from bovine spinal cord. The enzyme was purified about 230-fold from a concentrated spinal cord extract. Upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis, it stained as a protein of Mr 55,000. The purified enzyme is optimally active at around pH7 and has essential thiol groups. It appears to be highly specific for dynorphin B (Km = 11 microM) but not for alpha-neoendorphin or dynorphin A, two other opioids included in the prodynorphin precursor. From its specificity, molecular size, and inhibitory spectrum, this enzyme is different from other known dynorphin-converting or -degrading enzymes and appears to be a unique and novel endoprotease.
Different strategies for HPLC separation, including molecular sieving, ion-exchange, and hydrophobic interaction as well as reversed phase chromatography, were used to study molecular components in human cerebrospinal fluid (CSF). The separations were followed by photodiode-array UV detection, which is a recently developed technique allowing a direct and rapid discrimination between peptides and proteins differing in their content of aromatic amino acids. By the various HPLC techniques in conjunction with diode-array detection it was possible to identify and characterize several protein and peptide components present in CSF. The procedure also allowed quantitative analysis of CSF proteins using minute amounts of the fluid.
We measured plasma and cerebrospinal fluid (CSF) beta-casomorphin-8, a product of beta-casein hydrolysis which has opioid activity, by RIA in women during late pregnancy and lactation and in nonpregnant nonpuerperal women. Before RIA, the samples were acidified and extracted by reverse phase silica gel chromatography, which removed most of the beta-casein. Lactating women had a significantly higher mean plasma beta-casomorphin-8 concentration (2.66 nmol/L; n = 8) than women in late pregnancy (0.82 nmol/L; n = 8) and nonpregnant women (0.32 nmol/L; n = 5). The CSF beta-casomorphin-8 concentration also was significantly higher in lactating women (mean, 0.35 nmol/L; n = 8) than during late pregnancy (0.22 nmol/L; n = 8) or in nonpregnant women (0.15 nmol/L; n = 5). A positive correlation was found between plasma and CSF beta-casomorphin-8 levels in the entire study group. The milk beta-casomorphin-8 concentration, measured in five puerperal women, averaged 19.8 nmol/L. Thus, there is a decreasing concentration gradient between milk and plasma and between plasma and CSF. Chromatographic analysis revealed mol wt heterogeneity of the RIA-active material. In CSF at least three different components were detected, two of mol wt around 900-2,000 and one of approximately 12,000. One of the low mol wt components coeluted in several chromatographic systems with synthetic beta-casomorphin-8 (mol wt, 900). Such a component was not found in milk or plasma, in which the major activity was due to larger sized peptides. The major peaks in milk were around 1,500-2,000 and 12,000 mol wt, corresponding to the larger peaks in CSF. The results suggest that fragments of the milk protein beta-casein may cross the breast parenchyma-blood barrier into plasma and subsequently penetrate the blood-brain barrier to reach the central nervous system. Thus, mammary tissue may assume endocrine function during galactopoiesis, and beta-casein could be considered a prohormone.
Three putative processing enzymes, each with defined action in a prohormone system, a 'pro-ocytocin-neurophysin convertase' from bovine neurohypophysis secretory granules, a 'Leu-enkephalin Arg6 generating enzyme' from human CSF and the endoprotease from the 'S-28 convertase' complex of rat brain cortex, were tested for their ability to hydrolyze peptides deriving from pro-ocytocin, pro-enkephalin B and pro-somatostatin, respectively at pairs of basic amino acids. The observations suggest that structural parameters specified by the peptide region around the dibasic moieties govern recognition by the enzyme and define which peptide bond is hydrolyzed.
Defatted breast milk from women with postpartum psychosis and from healthy lactating women was analyzed by high-resolution gel permeation chromatography as well as by sodium dodecyl sulfate (SDS) and urea polyacrylamide gel electrophoresis. The gel permeation procedure allowed quantitative analysis of milk proteins (including beta-casein) with minute amounts of defatted milk (10-15 microliter). By electrophoresis, further characterization of the protein pattern, including the beta-casein fraction, was obtained. Milk samples from five control and seven psychotic subjects were analyzed. The concentration of the beta-casein-containing peak was significantly lower in milk samples from the psychotic group by both chromatography and electrophoresis. These lower levels of beta-casein may result from a higher rate of enzymatic degradation generating i.a. peptides with opioid activity, as shown earlier in plasma and CSF of women with postpartum psychosis.
This paper describes the enzyme catalyzed synthesis of the undecapeptide substance P from its non-associated fragments (1-7) and (8-11) or (1-8) and (9-11). The fragment condensation was mediated by the use of product specific antibodies as molecular traps. As catalyst a previously purified endopeptidase was used which specifically hydrolyzes substance P at the Phe7-Phe8 and Phe8-Gly9 bonds. The synthesis was performed in analytical scale and product formation was guided by reversed phase HPLC combined with radioimmunoassay. It appeared that the substance P fragments (1-8) and (9-11) were condensed to a larger extent than (1-7) and (8-11). This observation may well result from the higher affinity of the antibodies observed for substance P (8-11) as compared to that found for the other fragments. Increased concentration of the antibodies also seemed to result in enhanced resynthesis of substance P.
Endogenous opioid systems may be altered as a consequence of addiction, but evidence to support this idea is meager so far. We obtained 136 cerebrospinal fluid (CSF) samples from 72 opioid addicts during four distinct states: methadone maintenance, detoxification from methadone, opioid antagonist treatment, and drug-free status. CSF endorphins were measured in 86 patients samples using a radioreceptor assay (RRA), and beta-endorphin levels were measured in 85 patient samples using a radioimmuno assay (RIA). During detoxification, both RRA fraction I and beta-endorphin showed a generally similar pattern of changes. Both were lowest when measured 40-50 hr after the last opioid dose, and both showed an apparent rebound to higher than methadone maintenance values at 60-70 hr following the last dose. During methadone maintenance and drug-free states, the addicts' levels of fraction I RRA endorphins in the CSF were higher than levels found in a normal control group. Fraction II endorphins were also elevated in the addicts who were drug free. In contrast, CSF beta-endorphin during both methadone maintenance and drug-free states was lower in the addicts as compared to the normal, drug-naive group. Except for the pattern found during detoxification, there were no consistent changes in endorphin levels across different states of addiction.
Peptides with hormonal or neuronal activity are derived by enzymatic processing from pro-hormones, which by themselves are biologically inert. Processing and other enzymatic conversions may occur step-wise, leading to the formation of a cascade of biologically active (or inactive) peptides. The neurokin in substance P is known to be metabolically transformed both by amino- and endopeptidases. More N-terminal substance (1-7) has been found than C-terminal (2-11 to 5-11) fragments in various CNS areas. The substance P (1-7) fragment also shows biological activity e.g., providing analgesia, lowering blood pressure, inhibiting aggressive behavior and (in contrast to substance P) inhibiting grooming behavior. An endopeptidase generating substance P (1-7) and to a lesser extent, substance (1-8), has been isolated and characterized from human cerebrospinal fluid (CSF) and bovine spinal cord, as a metalloenzyme with essential SH-groups. Substance P co-exists with calcitonin gene related peptide (CGRP) in a large population of non-myelinated primary afferent ('pain') fibers. Intrathecal injection of substance P causes behavioral and physiological responses which are potentiated and prolonged by CGRP. It was found that CGRP competes with substance P for the endopeptidase. It is suggested that the main action of CGRP in the spinal cord is to inhibit substance P degradation.