Phosphorylation of Delta Sleep-Inducing Peptide (DSIP) by casein kinase II in vitro

A phosphorylated analogue of DSIP at Ser⁷ has been shown to exist endogenously by immunochemical studies. An enzyme which could phosphorylate DSIP has not yet been identified. In the present study, we examined DSIP as a substrate for in vitro phosphorylation by casein kinase II. DSIP was phosphorylated by the enzyme with apparent K_m and V_max values of 20 mM and 90.9 nmol/min/mg protein, respectively. Both ATP and GTP were utilized as phosphoryl donors. Phosphorylation of DSIP was inhibited by heparin and enhanced by spermine. These results demonstrate that DSIP can serve as a possible substrate for casein kinase II in vitro.     

Interaction of delta sleep-inducing peptide and its analogues with cellular membranes: A structure-function analysis

The possibility of a correlation between the membrane properties of the delta sleep-inducing peptide (DSIP) and its analogues and their biological activity in vivo was examined by a comparative study of the membrane effects of these peptides. The peptides exhibiting biological activity in vivo were shown to cause a statistically reliable disordering of lipids in thrombocyte plasma membranes similar to the effect of DSIP. The membrane effect of the D-Val²-, D-Tyr²-, and Tyr¹, Pro² analogues of DSIP had the same bimodal dose dependence characteristic of natural DSIP. Only a slight nonspecific lipid disordering was registered for Trp-Asp-Ala-Ser-Gly-Glu, a biologically inactive hexapeptide analogue. These results indicate a correlation between the biological activity of the peptides during in vivo tests and their membrane properties in vitro. The structure-function relationship was studied within the group of DSIP analogues examined in vitro. The DSIP modeling effect, especially pronounced under the action of stress factors, was suggested to be directly associated with the ability of DSIP to change the dynamic structure of biological membranes.     

Amphetamine-induced locomotor behavior of mice is influenced by DSIP

The delta sleep-inducing peptide (DSIP) has been shown to induce effects other than only delta sleep. One of these effects was the paradoxical thermoregulatory and locomotor response of rats to amphetamine after DSIP administration. In the present investigation we found similar effects of DSIP on the locomotor activity in mice. However, two different doses of DSIP (30 and 120 nmol/kg) and 3 doses of amphetamine (4, 10, and 15 mg/kg) produced a complex pattern of effects in mice tested at 22 degrees C. In general, DSIP-treated mice showed lower locomotor activity after amphetamine than controls, but under two conditions, both using 15 mg/kg amphetamine, DSIP produced higher scores; this occurred in the first two hours after amphetamine for the 30 nmol/kg DSIP group and in the third hour for mice given 120 nmol/kg DSIP. The results indicate that the effects of DSIP on locomotor behavior were dependent on the dosage of the peptide and the time of measurement as well as the level of amphetamine stimulation.     

δ-睡眠肽与GFP融合蛋白的表达和纯化

构建δ-睡眠肽(DSIP)蛋白与GFP的融合基因表达载体,高效表达和纯化GFP-DSIP融合蛋白。通过SOE-PCR拼接DSIP全长编码基因,并使得DSIP上游具有肠激酶识别位点,经双酶切定向克隆至表达载体pET-28a,构建重组载体pET-28a-DSIP,通过PCR扩增GFP全长编码基因,经双酶切定向克隆至pET-28a-DSIP,构建原核重组表达载体pET-28a-GFP-DSIP,通过双酶切和测序鉴定后,导入E.coli BL21宿主菌中,IPTG诱导表达融合蛋白,采用镍亲和层析和分子筛凝胶层析获得高纯度蛋白,SDS-PAGE分析鉴定。经测序鉴定成功构建了原核重组表达载体pET-28a-GFP-DSIP,在IPTG诱导下获得可溶性的绿色荧光蛋白与睡眠肽的融合蛋白,经Ni-NTA亲和层析纯化成功获得高纯度的融合蛋白。成功构建了DSIP与GFP融合基因的重组表达载体,确定了GFP-DSIP融合蛋白诱导表达的最佳条件,获得了较高纯度的融合蛋白,为进一步研究DSIP蛋白的生物学功能奠定了基础。     

Delta sleep-inducing peptide (DSIP): a still unresolved riddle

Delta sleep-inducing peptide (DSIP) was isolated from rabbit cerebral venous blood by Schoenenberger-Monnier group from Basel in 1977 and initially regarded as a candidate sleep-promoting factor. However, the link between DSIP and sleep has never been further characterized, in part because of the lack of isolation of the DSIP gene, protein and possible related receptor. Thus the hypothesis regarding DSIP as a sleep factor is extremely poorly documented and still weak. Although DSIP itself presented a focus of study for a number of researchers, its natural occurrence and biological activity still remains obscure. DSIP structure is different from any other known representative of the various peptide families. In this mini-review we hypothesize the existence of a DSIP-like peptide(s) that is responsible (at least partly) for DSIP-like immunoreactivity and DSIP biological activity. This assumption is based on: (i) a highly specific distribution of DSIP-like immunoreactivity in the neurosecretory hypothalamic nuclei of various vertebrate species that are not particularly relevant for sleep regulation, as revealed by the histochemical studies of the Geneva group (Charnay et al.); (ii) a large spectrum of DSIP biological activity revealed by biochemical and physiological studies in vitro; (iii) significant slow-wave sleep (SWS) promoting activity of certain artificial DSIP structural analogues (but not DSIP itself!) in rabbits and rats revealed by our early studies; and (iv) significant SWS-promoting activity of a naturally occurring dermorphin-decapeptide that is structurally similar to DSIP (in five of the nine positions) and the sleep-suppressing effect of its optical isomer, as revealed in rabbits. Potential future studies are outlined, including natural synthesis and release of this DSIP-like peptide and its role in neuroendocrine regulation.     

Regulation by Delta-Sleep-Inducing Peptide of the Neurochemical Changes in the Brain Associated with Dopaminergic System Hyperactivity

The influence of a single injection of delta-sleep-inducing peptide (DSIP; 30 g/kg body weight) on neurochemical parameters of rats' brain was studied under the conditions of chronic administration of dopamine analogs inducing DA-system hyperactivity – 50 mg/kg body weight of L-DOPA for 30 days or 2,5 mg/kg body weight of amphetamine for 21 days. The parameters of serotonergic system (MAO A activity, 5-HT, and 5-HIAA contents) and of dopaminergic system (MAO B activity, DA, NA, and HVA contents) were investigated in the cortex and caudate nucleus of control, DA or amphetamine, and DSIP receiving rats. Changes caused by the two DA-system activating drugs had both similarities and differences, and the corrective action of DSIP also had certain peculiarities depending on the pharmacological preparation used for the induction of DA-system hyperactivity and on the investigated brain structure. It is supposed that DSIP action might be based on the activation of serotonergic system that ensures the adaptive behavior of the animals.     

Plasma levels of DSIP in infants in the first year of life and SIDS risk.

In searching for abnormalities related to the sudden infant death syndrome (SIDS), delta sleep-inducing peptide (DSIP), a regulatory peptide with sleep promoting actions, was investigated in the first year of life in four groups of children: (1) preterm infants (n = 28), (2) infants with a high mean apnea duration evaluated polysomnographically (n = 26), (3) healthy full-term infants (n = 37) and (4) siblings of SIDS-victims (n = 26). DSIP was radioimmunoassayed in plasma. Half of the infants were also investigated polygraphically during sleep. The ratio between quiet sleep and active sleep was determined. There was no age dependence of the plasma level of DSIP in the first year of life but there was an increase in the ratio of quiet/active sleep depending of maturity. The level of DSIP in healthy full-term infants was significantly higher (P less than 0.05) (median: 1885 pmol/l, interquartile range: 757 pmol/l) than in preterms (1595; 385) and in infants with a high mean apnea duration (1542; 373). There was no significant difference in DSIP concentrations between healthy full-term infants and SIDS-siblings (1605; 271).     

Delta Sleep-Inducing Peptide Modulates the Stimulation of Rat Pineal N-Acetyltransferase Activity by Involving the α1 -Adrenergic Receptor

Delta sleep-inducing peptide (DSIP) has been isolated and characterized by its capacity to enhance delta sleep in rabbits. Up to now, sleep was the main target of DSIP research, but different extra-sleep effects of the peptide have been reported as well. Several mechanisms of action have been proposed, though no convincing evidence for any of them has been obtained so far. We recently detected that DSIP reduced the nocturnal increase of N-acetyltransferase (NAT) activity in rat pineal in a dose-dependent manner. The activity of this enzyme is known to be induced by adrenergic agonists and several studies have suggested that stimulation of alpha 1-adrenergic receptors potentiates the "basic" effect of beta-receptors. DSIP in the range between 20 and 300 nM significantly enhanced NAT activity induced by 10(-6) M norepinephrine in vitro, and a similar effect was observed with 2nMP-DSIP, a phosphorylated analog. Incubation with prazosin eliminated the enhancement, whereas propranolol reduced norepinephrine stimulation that was still increased by P-DSIP and probably DSIP. It was concluded that the sleep-peptide and its analog modulate the alpha 1-adrenergic receptor of rat pineal in its response to adrenergic agonists. The same mechanism may also be responsible for other biological activities of DSIP such as sleep-induction and stress-tolerance.     

Characterization of Delta-Sleep-Inducing Peptide-Evoked Release of Met-Enkephalin from Brain Synaptosomes in Rats

Delta-sleep-inducing peptide (DSIP) stimulates the release of Met-enkephalin (Met-ENK) from superfused slices of the rodent lower brainstem in vitro. In our present study, DSIP (10(-10)-10(-9) M) induced a significant release of Met-ENK from medullary synaptosomes of rats. This DSIP-evoked release of Met-ENK was Ca2+ dependent and tetrodotoxin (TTX) insensitive. Furthermore, DSIP (10(-11)-10(-9) M) significantly increased 45Ca2+ uptake in medullary synaptosomes. These results demonstrate that DSIP acts directly on the nerve endings of Met-ENK-containing neurons to release this pentapeptide by generating a Ca2+ influx into these neurons. Effects of DSIP on Met-ENK release in other discrete brain regions were also studied. Significant DSIP-evoked Met-ENK release from synaptosomes was observed in the cortex, hypothalamus, and midbrain (at concentrations of 10(-10) and 10(-9) M) and in the hippocampus and thalamus (only at 10(-9) M), but not in the striatum. In the hypothalamus, the release of Leu-enkephalin from its synaptosomes was slightly, but not significantly, enhanced by DSIP (10(-10)-10(-8) M). Our findings demonstrate that DSIP triggered a Ca2+ influx in nerve endings to induce a subsequent release of Met-ENK from neurons in only certain brain regions.     

Regulation of Free Radical Processes by Delta-Sleep Inducing Peptide in Rat Tissues under Cold Stress

An intraperitoneal injection of an exogenous delta-sleep inducing peptide (DSIP) at a dose of 12 g/100 g body weight shifted the prooxidant–antioxidant balance of free radical process (FRP) in tissues and erythrocytes of rats: the activities of antioxidant enzymes (superoxide dismutase, catalase, glutathione peroxidase, and glutathione reductase) and the concentrations of antioxidants (reduced glutathione in particular) increased. The DSIP stimulated the myeloperoxidase activity in blood neutrophils and had no effect on the activity of xanthine oxidase, a prooxidant enzyme, in the brain and liver. Cold stress displaced the prooxidant–antioxidant balance by increasing the xanthine oxidase activity in tissues and decreasing the myeloperoxidase activity in blood neutrophils; it also inhibited the enzyme antioxidant activities in tissues and erythrocytes that was neutralized by an increased ceruloplasmin activity in blood plasma and by an elevated level of antioxidants in rat blood and tissues. Preliminary administration of DSIP to animals exposed to cold stress restored the prooxidant–antioxidant balance: it normalized the myeloperoxidase activity in blood neutrophils, decreased the xanthine oxidase activity, and increased the activity of antioxidant enzymes in tissues and erythrocytes restoring the antioxidant level. The molecular regulation mechanism of free radical processes by DSIP in tissues under stressful conditions is discussed.     

JmjC-domain-containing histone demethylases of the JMJD1B type as putative precursors of endogenous DSIP

Delta sleep inducing peptide (WAGGDASGE, DSIP) is a well known multifunctional regulatory peptide. Numerous studies have confirmed its stress-protective and adaptive activity which is independent of the origin or nature of the stress or other harmful factors. However, the biosynthetic origin of DSIP remains obscure, since nothing is known of its protein precursor(s) and their encoding gene(s). We have performed a comprehensive analysis of available gene and protein databases for homologous peptide sites within mammalian resources including man. A family of Jumonji C (JmjC)-domain-containing histone demethylases was shown to contain a sequence fragment closely homologous to DSIP. One type of these ubiquitous and phylogenetically ancient proteins encoded by JMJD1B gene includes the WKGGNASGE sequence that differs from DSIP by only 2 amino acid residues in positions 2 and 5. The respective peptide was synthesized and its biological effects were evaluated in a preliminary way in the forced swimming and antitoxic tests. We suggest that the histone demethylases of the JmjC-group containing DSIP-related region can be considered as possible protein precursors of endogenous peptides with DSIP-like activity.     

DSIP affects adrenergic stimulation of rat pineal N-acetyltransferase in vivo and in vitro

The natural occurrence, sleep, and extra-sleep effects of delta sleep-inducing peptide (DSIP) have been shown by different laboratories. However, neither an in vitro assay system nor a probable mechanism of action of the peptide have been conclusively demonstrated so far. The recent finding that DSIP influences the nocturnal rise of N-acetyltransferase (NAT) activity in rat pineal led us to investigate a possible effect on pharmacologically induced NAT activity in vivo and in vitro. Stimulation of the enzyme with adrenergic drugs such as isoproterenol and phenylephrine was reduced by DSIP at doses of 150 and 300 μg/kg injected subcutaneously. In vitro, 6, 150 and 300 nM DSIP attenuated isoproterenol stimulation of the enzyme in cultured pineals, whereas 150 nM DSIP effectively reduced stimulation induced by a combination of the two drugs. The peptide alone did not influence NAT activity in vitro, but produced a slight stimulation in vivo. To our knowledge, these results represent the first report of a direct interaction of DSIP with adrenergic transmission. The in vitro system could prove useful for establishing possible mechanism(s) of action of the ‘sleep peptide.’     

Delta sleep-inducing peptide (DSIP)-like material is absorbed by the gastrointestinal tract of the neonatal rat

Entry of delta sleep-inducing peptide (DSIP) into the circulation from the gastrointestinal (GI) tract was studied in unweaned rat pups. The pups were fed an analog of DSIP (N-Tyr-DSIP) or 125I-N-Tyr-DSIP and blood samples collected. Significant increases in plasma DSIP-like immunoreactivity occurred after the feeding of 100 ωg/animal of N-Tyr-DSIP but not after vehicle (normal saline) or 1 ωg/animal. Column chromatography showed this immunoreactivity to coelute with intact DSIP and des-Trp¹- DSIP. A small but statistically significant increase of immunoreactivity occurred in the plasma of pups whose nursing mothers were injected with N-Tyr-DSIP but not in those whose mothers were injected with saline. Radioactivity appeared in both the brain and blood of 1–2 and 10 day old rat pups fed 125I-N-Tyr-DSIP. Although only a small amount of the radioactivity in plasma co-eluted with intact 1251I-N-Tyr-DSIP on column chromatography, almost all of the radioactivity in brain did, suggesting that the radioactivity in the brain represented crossing of the blood-brain-barrier by the peptide and not just contamination by blood. The results cannot be explained by either regurgitation of intestinal contents, or by stimulation of endogenous peptide. They show that a DSIP peptide administered orally can be absorbed through the GI tract into the systemic circulation.     

Passage of delta sleep-inducing peptide (DSIP) across the blood-cerebrospinal fluid barrier

Unidirectional flux of 125I-labeled DSIP at the blood-tissue interface of the blood-cerebrospinal fluid (CSF) barrier was studied in the perfused in situ choroid plexuses of the lateral ventricles of the sheep. Arterio-venous loss of 125I-radioactivity suggested a low-to-moderate permeability of the choroid epithelium to the intact peptide from the blood side. A saturable mechanism with Michaelis-Menten type kinetics with high affinity and very low capacity (approximate values: Kt = 5.0 +/- 0.4 nM; Vmax = 272 +/- 10 fmol.min-1) was demonstrated at the blood-tissue interface of the choroid plexus. The clearance of DSIP from the ventricles during ventriculo-cisternal perfusion in the rabbit indicated no significant flux of the intact peptide out of the CSF. The results suggest that DSIP crosses the blood-CSF barrier, while the system lacks the specific mechanisms for removal from the CSF found with most, if not all, amino acids and several peptides.     

Saturable mechanism for delta sleep-inducing peptide (DSIP) at the blood-brain barrier of the vascularly perfused guinea pig brain

Cellular uptake of [125I] labelled DSIP at the luminal interface of the blood-brain barrier (BBB) was studied in the ipsilateral perfused in situ guinea pig forebrain. Regional unidirectional transfer constants (Kin) calculated from the multiple-time brain uptake analysis were 0.93, 1.33 and 1.66 microliter.min-1 g-1 for the parietal cortex, caudate nucleus and hippocampus, respectively. In the presence of 7 microM unlabelled DSIP the brain uptake of [125I]-DSIP (0.3 nM) was inhibited, the values of Kin being reduced to 0.23-0.38 microliter.min-1 g-1, values that were comparable with the Kin for mannitol. The rapidly equilibrating space of brain, measured from the intercept of the line describing brain uptake versus time on the brain uptake ordinate, Vi, was greater for [125I]-DSIP than for mannitol; in the presence of unlabelled DSIP this was reduced to that of mannitol, and it was suggested that the larger volume for [125I]-DSIP represented binding at specific sites on the brain capillary membrane. L-tryptophan, the N-terminal residue of DSIP, in concentrations of 7 microM and 1 mM, inhibited Kin without affecting Vi. A moderate inhibition of Kin was obtained by vasopressin ([Arg8]-VP), but only at a concentration as high as 0.2 mM. The results suggest the presence of a high affinity saturable mechanism for transport of DSIP across the blood-brain barrier, with subsequent uptake at brain sites that are highly sensitive to L-tryptophan, and may be modulated by [Arg8]-VP.     

Extraction and immunochemical characterization of delta sleep-inducing peptide-like material from the porcine pituitary and adrenal gland

The naturally occurring forms of delta sleep-inducing peptide (DSIP) are not fully identified. In the present study, porcine pituitaries and adrenal glands were extracted in water, saline or acid under various conditions and immunoreactive DSIP (IR-DSIP) quantified by radioimmunoassay. The highest concentrations were measured in anterior pituitary extracts (40.8 +/- 2.6 ng/g tissue weight) recovered using water with aprotinin. However, high performance liquid chromatography (HPLC) indicated degradation of hydrophobic forms of IR-DSIP in water extracts. Extraction in acetic acid including C18 Sep-Pak purification resulted in an elution profile of IR-DSIP in adrenal extracts with a major peak coeluting with synthetic DSIP [DSIP(1-9)], whereas anterior pituitary extract showed material of higher hydrophobicity. Approximately 30% of IR-DSIP in anterior pituitary as well as in adrenal gland extracts seemed to be glucosylated, as based on concanavalin A chromatography. One of the DSIP-immunoreactive components by immunoblotting (molecular mass 25 kDa) was identified in both pituitary and adrenal gland extracts. In conclusion, several chromatographically distinct forms of IR-DSIP are present in the porcine pituitary and adrenal gland. IR material eluting as DSIP(1-9) is present in adrenal gland extract. The procedure and solution used for tissue extraction seem to be essential in order to obtain reliable elution positions on HPLC.     

Sleep-Inducing Properties of DSIP Analogs: Structural and Functional Relationships

The sleep-inducing activity of Delta Sleep-Inducing Peptide (DSIP) and its 13 synthetic analogs has been studied on rabbits with preliminary implanted electrodes. The peptides were injected into the lateral ventricle of cerebrum. Polygraphic computer monitoring of sleep–wake states was carried out at daytime for 7–12 h. DSIP and most analogs had no statistically significant effect on sleep compared to the control administration of saline to the same animals. [NMeAla²]DSIP and [Pro²]DSIP had a pronounced sleep-inducing effect and reliably increased the proportion of slow-wave sleep by 10–15% on average compared to the control. Several other analogs had a week sleep-inducing effect, increasing the proportion of slow-wave sleep during specific recording time only. [-Ala²]DSIP significantly suppressed sleep. In addition, this analog, as well as parent DSIP and four proline-containing nonapeptides, slightly increased the body temperature. The revealed differences may be due to both conformation properties and proteolytic resistance of the studied molecules, and it may reflect their indirect involvement in the control sleep–wake hormonal processes.     

Delta-sleep inducing peptide (DSIP) and its fragments as the modulators of neural transmission in the central nervous system

The present study is a continuation of our previous experiments on DSIP activity which have revealed that nonapeptide DSIP inhibits hippocampal electrical activity of the 4-7 c/s frequency band. The aim of the present study was to find which of the known DSIP fragments is responsible for its activity, i.e. to find the active site of the molecule. The experiments were carried out with the entire DSIP molecule and its three different fragments. The method of threshold continuous arousal pattern (TCAP) monitoring was used as the indicator of DSIP activity. It was found that the entire DSIP molecule increased TCAP, while its 1-5 fragment decreased it 1-4 and 5-9 fragments had no noticeable effect.     

Delta sleep inducing peptide (DSIP): effect on respiration activity in rat brain mitochondria and stress protective potency under experimental hypoxia

Neuromodulatory delta sleep inducing peptide (DSIP) seems to be implicated in the attenuation of stress-induced pathological metabolic disturbances in various animal species and human beings. Mitochondria, as cell organelles, are considered especially sensitive to stress conditions. In this work, the influence of DSIP and Deltaran((R))-a recently developed product based upon DSIP-on processes of oxidative phosphorylation and ATP production in rat brain mitochondria and rat brain homogenates was studied. A polarographic measurement of oxygen consumption was applied to evaluate the impact of DSIP on maximal rates of mitochondrial respiration and coupling of respiration to ATP production. We provide evidence that DSIP affected the efficiency of oxidative phosphorylation on isolated rat brain mitochondria. This peptide significantly increased the rate of phosphorylated respiration V3, while the rate of uncoupled respiration V(DNP) remaining unchanged. It enhanced the respiratory control ratio RCR and the rate of ADP phosphorylation. DSIP and Deltaran exhibited the same action in rat brain homogenates. We also examined the influence of DSIP under hypoxia when mitochondrial respiratory activity is altered. In rats subjected to hypoxia, we detected a significant stress-mediated reduction of V3 and ADP/t values. Pretreatment of rats with DSIP at the dose of 120 microgram/kg (i.p.) prior to their subjection to hypoxia completely inhibited hypoxia-induced reduction of mitochondrial respiratory activity. The revealed capacity of DSIP to enhance the efficiency of oxidative phosphorylation found in vitro experiments could contribute to understanding pronounced stress protective and antioxidant action of this peptide in vivo.     

Effect of delta sleep-inducing peptide, anticonvulsant preparations and nicotinamide on generalized seizure activity

The influence of delta-sleep inducing peptide (DSIP) upon seizures induced by corazol, bicuculline, picrotoxin, strychnine, thiosemicarbazide were investigated in experiments on F1(CBA X C57 BL/6) mice. It was shown that DSIP increased the latency of first seizure manifestation which were induced by corazol, bicuculline and picrotoxin and also resulted in a suppression of seizure severity of corazol and bicuculline induced seizures. Anticonvulsant action of DSIP was evident under the condition of the mild severity seizures development. The effect of DSIP was mostly pronounced in range of its doses from 10 to 100 mcg/kg. DSIP when combined with phenobarbital, carbamazepine, diphenylhydantoin or nicotinamide enhanced the antiepileptic effects of these anticonvulsant drugs.