Immunochemical characterization of neurotensin-like peptides in chicken

Using radioimmunoassay and 3 region specific antisera toward bovine neurotensin (NT), the NT-like peptides in chicken have been shown to differ from NT but to strongly resemble its COOH-terminal region. Three substances were identified, one of which resembled NT biologically and appeared to share 7 or 8 of its COOH-terminal residues. The two other peptides were smaller than NT but seemed to possess 4–6 residue homologies with it. Tissue distribution studies indicated that the chicken pancreas and thymus had unusually high levels of this material (>200 fold that in rat) and that the 3 substances were distributed differently in tissues. Chromatographic studies showed that the peptides obtained from brain, intestine, thymus, and pancreas were similar. These results, demonstrating evolutionary conservation of the COOH-terminal region of NT, are in keeping with the known importance of this region for biological activity. These findings also suggest the existence of an NT-family of peptides serving multiple biological roles.     

Immunoreactive neurotensin and somatostatin in the chicken thymus

Summary Two distinct populations of endocrine cells in the chicken thymus display neurotensin and somatostatin immunoreactivity, respectively. Both cell types are few in number at hatching but proliferate rapidly during the first week. The neurotensin cells are Grimelius-positive and Hellerström-Hellmannegative. The somatostatin cells are Grimelius-negative and Hellerström-Hellman-positive. Both cell populations are non-argentaffin. The somatostatin-like material extracted from chicken thymus behaves immunochemically and chromatographically similar to synthetovine somatostatin, while the neurotensin-like material, from the thymus as well as from the gut, differs from synthetic bovine neurotensin in that it appears larger in size and more basic.     

Binding and biologic activity of neurotensin in guinea pig ileum

Experiments were performed to relate receptor binding to biologic activity for the contractile effect of neurotensin (NT) in guinea pig ileum. The contractile response was examined on pieces of ileum under 1 g tension in a 5 ml bath of oxygenated Tyrode's at 38°C. NT contracted the longitudinal muscle (ED₅₀, 0.3 nM), the 2–3 g response peaking at 1 min and fading rapidly. In the presence of atropine (1 μM), ≥50% of the response was blocked and the residual effect gave an ED₅₀ of 1.4 nM. In the presence of atropine and CP-96,345, a substance P receptor antagonist (0.2 μM), no contraction was observed at 20 nM NT. Thus, there were two components to the response, one involving acetylcholine (ED₅₀, 0.3 nM) and one substance P (ED₅₀, 1.4 nM). Using membrane preparations and ¹²⁵I-labeled NT, specific, high affinty receptors for NT were demonstrated in the muscle and myenteric plexus. Scatchard analyses indicated the presence of two binding sites (K_ds: 0.1 nM and 2 nM). Sodiu ion and GTP analogs inhibited binding. Binding and biologic activity were similar in regard to dependence on specific groups within NT and sensitivity to metal ions. The high potency of Hg⁺⁺ was consistent with an involvement of free sulfhydryl group(s) in the binding reaction; this was supported by work with SH-directed agents. The results suggest that two receptor types or configurations may mediate the two components of the contractile effect of NT on guinea pig ileum.     

Radioimmunoassay for Lys8, Asn9, neurotensin 8–13: Tissue and subcellular distribution of immunoreactivity in chickens

A sensitive and specific radioimmunoassay (RIA) for Lys⁸, Asn⁹, neurotensin 8–13 (LANT-6) has been developed which utilizes ¹²⁵I-labeled LANT-6 and rabbit antisera raised towards conjugates of synthetic LANT-6 and bovine thyroglobulin. The antiserum described (TG-22) allows the detection of ca 100 fmol of LANT-6 and crossreacts <0.01% with chicken or bovine NT. Dose-response relationships for the native (chicken) and synthetic peptides were indistinguishable. Using this assay the distribution of immunoreactive LANT-6 (iLANT-6) through various tissues of the chicken was studied and compared with that of chicken NT (iNT) determined by RIA. Both iNT and iLANT-6 were found primarily in the brain and gastrointestinal tract, however, their regional distributions were found to differ. Subcellular distribution studies in homogenates of chicken brain indicated that obth iNT and iLANT-6 were associated with synaptosome-like and vesicle-like particles. In homogenates of small intestine, pancreas and colon iNT and iLANT-6 appeared to be within osmotically sensitive, sedimentable particles. Analyses using high pressure liquid chromatography established that chicken iLANT-6 co-eluted with the synthetic peptide and that similar substances were present in extracts of rat brain and intestine. These results are consistent with “messenger” roles for these peptides.     

A common precursor to neurotensin and LANT6 and its differential processing in chicken tissues

Antisera towards neurotensin (NT) and the structurally related peptide, LANT6, were used to characterize immunoreactive peptides and proteins in extracts of chicken tissues. A 17 kDa protein was identified by Western blotting as a potential precursor to NT and LANT6. However, the posttranslational processing of this common precursor appeared to be tissue specific, giving rise to disproportionate amounts of NT and LANT6, along with varying expression of a large molecular LANT6 (M_r, 15 kDa). The intestinal cells containing immunoreactive NT, LANT6, and large molecular LANT6 behaved similarly during fractionation by size and density. These activities also banded together in particles resembling vesicles during centrifugation of isotonic homogenates of tissue. These results suggest that chicken NT and LANT6 are biosynthesized as parts of the same precursor, the processing of which can give rise to a variety of products stored within secretory vesicles.     

Distribution and immunochemical character of neurotensin-like material in representative vertebrates and invertebrates: Apparent conservation of the COOH-terminal region during evolution

Using a radioimmunoassay for bovine neurotensin (NT) and various region specific antisera which react selectively with different portions of the molecule, the presence of immunoreactive NT (iNT) in a wide range of vertebrate and invertebrate species has been demonstrated. While antisera directed towards the N-terminal region of NT recognized only mammalian forms of NT, antisera directed towards the C-terminal region of NT recognized materials from all species examined, including representatives of all vertebrates and invertebrate classes. When extracts of the brain and gut of the vertebrates examined were chromatographed on Sephadex G-25 multiple NT-like substances were observed, and the patterns of iNT obtained seemed to fall into three groups: (a) mammals, (b) birds and reptiles and (c) amphibians and fish. Extracts of invertebrates also exhibited multiple peaks of iNT on Sephadex G-25 and the profiles observed resembled those for lower vertebrates. Partially purified iNT obtained from chicken, turtle, dogfish and lobster was shown to increase hematocrit and induce cyanosis in anesthetized rats. These findings indicate (a) that NT-like substances appear to be present throughout the animal kingdom, (b) that the C-terminal region of NT is highly conserved while the N-terminal region varies, and (c) that in any one animal multiple substances sharing C-terminal homologies with NT exist. These findings are consistent with the notion that NT and related peptides participate in important processes basic to animal life and that their functioning depends highly upon elements located in their C-terminal regions. They further suggest the existence of an entire family of NT-related peptides in each animal form, possibly distributed differently and functioning differently in the various organs of the animal.     

Immunohistochemical localization of neurotensin in endocrine cells of the gut

Summary Endocrine cells displaying neurotensin immunoreactivity are found scattered in the jejuno-ileum of all mammals studied, including man. They are rather scarce in rat, guinea pig, rabbit and pig and fairly numerous in cat, dog and man. In most mammals the neurotensin cells predominate on the villi. Only in the dog are they more numerous in the crypts. In the chicken, neurotensin cells occur all along the intestinal tract. They are particularly numerous in the zone that joins the gizzard with the duodenum. The ontogeny of the neurotensin cells in the gut was studied in rats and chickens. In the rat, the cells are first observed in the jejuno-ileum immediately before birth. The adult frequency is reached 4–5 days later. In the chicken, neurotensin cells first appear in the colon in the 18 day old embryo and in the small intestine two days later (i.e. one or two days before hatching). A few days after hatching, the gut has achieved the adult number of neurotensin cells per unit area.     

Involvement of dopamine in the central effect of neurotensin on intestinal motility in rats

The effects of intracerebroventricular (ICV) administration of neurotensin (NT) before a meal on intestinal postprandial motility were examined in conscious rats chronically fitted with intraparietal Nichrome electrodes in the duodeno-jejunum. The effects were compared with those of two analogues, [D-Tyr¹¹]NT and [D-Trp¹¹]NT, resistant to degradation by brain peptidases. NT (10 μg ICV) delayed the occurrence of postprandial disruption of duodenal motility and blocked it on the jejunum. [D-Tyr¹¹]NT and [D-Trp¹¹]NT (1 μg ICV) elicited the same effects but at a ten-fold lower dose. NT administered peripherally just before a meal significantly lengthened the duration of the postprandial motor pattern. The central effect of NT on the fed pattern involved dopaminergic neurons as it was mimicked by dopamine, blocked by haloperidol and partly antagonized by either sulpiride or (+) SCH 23390. It is concluded that: 1) both D₁ and D₂ receptors are involved in the blocking effect of the postprandial disruption induced by central NT; 2) that [D-Tyr¹¹]NT and [D-Trp¹¹]NT are potent agonists at NT receptors in the brain.     

Isolation and quantitation of several new peptides from the canine neurotensin/neuromedin N precursor

Using antisera towards the bioactive peptides, neurotensin and neuromedin N, as well as towards the N-terminal and C-terminal regions of their shared 170-residue precursor, peptides representing various portions of the precursor were isolated from extracts of canine ileum. In total, seven peptides were isolated, two of which had not been previously identified. One was the C-terminal tail of the precursor (Gly-Ser-Tyr-Tyr-Tyr) and the other was the tail peptide extended N-terminally to include neurotensin (Glp-Leu-Tyr-Glu-Asn-Lys-Pro-Arg-Arg-Pro-Tyr-Ile-Leu-Lys-Arg-Gly-Ser-Tyr-Tyr-Tyr). By comparing the measured concentrations for each of the identified peptides, it was established that processing at the three Lys-Arg cleavage sites within the precursor did not occur to the same extent. Cleavage at the N-terminus of neuromedin N was 10% complete, that between neurotensin and the tail was 90% complete, and that between neuromedin N and neurotensin was 95% complete. Three immunoreactive proteins were also identified by immunochemical and chromatographic analyses: N-terminally extended neuromedin N (125 residues), N-terminally extended neurotensin (140 residues), and the entire 147-residue precursor. It was concluded that neurotensin, tail and large molecular neuromedin N were the primary products of processing for this precursor in canine ileum, while minor products included neuromedin N, neurotensin tail, and large molecular neurotensin.     

Isolation and characterization of rat vasoactive intestinal peptide

We have used gel filtration, ion exchange chromatography, affinity chromatography and reversed-phase HPLC to isolate vasoactive intestinal peptide from rat intestine. Microsequence analysis of 1 nmole peptide indicated that the sequence was identical to the porcine octacosapeptide VIP. In radioimmunoassay with four antisera and in the turkey pancreas bioassay, rat VIP was equipotent with highly purified preparations of porcine, human and canine VIP. A less basic rat VIP-variant was also isolated and the N-terminal decapeptide region that was sequenced was identical with that of porcine VIP.     

The Regional Distribution and Chromatographic Characterisation of Neurotensin-Like Immunoreactivity in the Rat Central Nervous System

Abstract: Carboxy- and amino-terminal specific neurotensin antisera have been characterized and used to determine the nature of neurotensin-like immunoreactivity in the rat central nervous system. Using these antisera, together with the separation of neurotensin-like immunoreactivity on reversephase HPLC columns, it is clear that the majority of rat central nervous system neurotensin-like immunoreactivity is indistinguishable from the synthetic tridecapeptide. However, smaller amounts of carboxy- and amino-terminal neurotensin-like immunoreactivity were detected, which may correspond to carboxy- and amino-terminal fragments of neurotensin. In addition, using the amino-terminal directed neurotensin antiserum, a detailed distribution of neurotensin-like immunoreactivity in the rat central nervous system is described. Highest amounts were found in the hypothalamus, central amygdaloid nucleus, bed nucleus of the stria terminalis and the substantia gelatinosa of the spinal cord and of the trigeminal region.     

肠道菌群影响黏膜屏障结构与功能的研究进展

肠道黏膜屏障具有防止致病性抗原侵入、维护肠道健康的功能。而肠道菌群是肠道黏膜屏障的重要构成部分,肠道菌群失调会导致肠道黏膜屏障的损伤,引起炎性肠病、肠易激综合征及肝、肾等多种疾病的发生发展。因此,本文从肠道黏膜的结构与功能及肠道菌群对其的影响等方面归纳总结肠道菌群对屏障系统的调控作用,从调节肠道微生态平衡、促进黏液分泌、影响紧密连接和肠道上皮通透性、激发肠黏膜免疫、调控肠上皮凋亡、影响肠上皮DNA稳定性及产生特殊代谢产物等方面阐述其作用机制,为临床胃肠道疾病及其并发症的治疗提供新的思路和方法。     

The stability and metabolism of intravenously administered neurotensin in the rat

The clearance and metabolism of synthetic and tritiated (³H) neurotensin (NT) were studied following its intravenous injection in a pharmacologic dose (500 pmol/kg) into anesthesized rats. Immunoreactive NT (iNT), measured in a radioimmunoassay (RIA) with use of a carboxyl-(C)-terminal directed antiserum, displayed an apparent half-life ($\text{t}\text{1}\text{2}$) of 0.55 min, while that measured by an amino-(N)-terminal directed antiserum had a $\text{t}\text{1}\text{2}$ of 5 min. The radiolabel from injected ³H-NT (³H on Tyr^3,11) had a $\text{t}\text{1}\text{2}$ of 6.5 min. High-pressure liquid chromatography of extracts of plasma obtained from the circulation 0.5–3 min after injection of NT and ³H-NT showed the presence of NT and the generation mainly of the fragments NT^1–8, NT^1–11, and NT^9–13, as well as free ³H-labeled tyrosine. The apparent half-lives of intravenously injected synthetic NT^1–8, NT^1–11 and NT^1–12 measured with the N-terminal RIA were 9, 5 and 5 min, respectively, while that for NT^9–13 was less than 0.5 min. These results indicate that exogenously injected NT is rapidly metabolized to form N-terminal fragments which are cleared more slowly than NT. These findings suggest that use of N-terminal antisera to detect the release of endogenous NT into the circulation is likely to yield measurements of the fragments NT^1–8 and NT^1–11 which thus far have been found to be biologically inactive.     

Isolation and primary structure of an amphibian neurotensin.

Using a radioimmunoassay system employing an antiserum which recognises the common C-terminal tripeptide (YIL) of neurotensin (NT) and neuromedin N (NN), immunoreactivity was identified in extracts of brain (65.8 pmol/g), small intestine (44.2 pmol/g) and rectum (13.2 pmol/g) of the European common frog (Rana temporaria). No immunoreactivity was detected in extracts of stomach and skin. Reverse-phase HPLC analysis of each tissue extract resolved a single immunoreactive peptide with identical retention time in each case. The immunoreactive peptide was isolated by reverse-phase HPLC from brain extracts and an N-terminal pyroglutamyl residue was successfully removed enzymatically. The molecular mass of des(pyroglutamyl) frog NT, determined by plasma desorption mass spectroscopy, was 1440 Da. The primary structure of this peptide was determined by gas-phase sequencing and the calculated molecular mass, 1440.7 Da, was in close agreement with that derived by mass spectroscopy. The full primary structure of frog NT was established as: QSHISKARRPYIL. When compared with bovine NT, frog NT exhibits five amino acid substitutions in the N-terminal region, whereas the C-terminal hexapeptide sequence (RRPYIL), which mediates the classical biological effects of NT, is completely conserved. Amphibia thus possess a tridecapeptide NT which is analogous to that of higher vertebrates and considerable constraints on the primary structure of the C-terminal biologically-active core have existed for a vast evolutionary time span.     

Organization of LHRH cells: differential apposition of neurotensin, substance P and catecholamine axons

A wealth of evidence suggests that catecholamines (particularly norepinephrine) influence gonadotropin secretion via a direct interaction with the LHRH neurons. Neuropeptides such as neurotensin (NT) and substance P (SP) are likewise implicated in the control of LHRH secretion, based on pharmacological and preliminary anatomical studies. Since sub-populations of LHRH neurons project to areas of the brain other than the median eminence, a detailed analysis of the topography of axonal interactions of catecholamines (CA), substance P and neurotensin with LHRH cells was conducted in adult male mice using dual immunocytochemical techniques. An analysis of the patterns of apparent contact of NT or SP axons on LHRH cells as determined by close apposition of immunoreactive axons to LHRH cells when viewed under a light microscope at high magnification revealed that the density of NT or SP axons was not a reliable index of the degree of contact; in many locations, NT and SP had similar densities yet a greater portion of the LHRH cells appeared contacted by SP than NT. NT axons were in close contact with up to one-third of the LHRH cells. Analysis of the location of these "contacted" cells did not reveal a discrete subnucleus controlled by NT. Rather, the NT-contacted cells were scattered throughout the LHRH cell field. Interactions of LHRH cells with SP axons were likewise uniform throughout most of the LHRH cell field, with the exception of the most anterior portion of the field. In the anterior septum, few SP axons appeared to contact LHRH cells. Elsewhere, most of the LHRH cells were in contact with SP axons. For the CAs, the fiber density in the regions of the LHRH cells was uniformly moderate, yet the pattern of cells contacted showed variation across the LHRH cell field, with most of the "contacted" cells located near the OVLT and medial preoptic area. These data suggest that LHRH cells may be differentially regulated by NT, SP and the CAs.     

活血化瘀法对肠道菌群结构及肠屏障功能影响的研究

随着人们对于肠道菌群种类以及作用认识的逐渐深入,我们发现作为人体庞大而又复杂的微生态系统,肠道菌群的结构及和菌群分布有紧密联系的肠屏障功能的改变与人体的健康息息相关。中药作为传统医学的一种治疗方法,其对人体的治疗作用是十分显著的,而活血化瘀法是使用具有消散作用及攻逐体内淤血作用的药物来治疗人类各种疾病的一种方法,这种方法对于肠道菌群以及肠屏障功能也产生了深远的影响。本文围绕肠道菌群结构改变及肠黏膜屏障功能的变化,对近十年关于肠道菌群与活血化瘀方药的相关文献进行综述,探究活血化瘀法（中药及中药复方）对于肠道菌群的影响,为临床治疗提供新思路。     

小碎斑鱼蛉幼虫肠道细菌分离及鉴定研究

目的从微生态学角度研究小碎斑鱼蛉幼虫的营养生理活动。方法从小碎斑鱼蛉幼虫肠道环境中进行分离、纯化和培养,获得3个细菌菌株,对其菌体形态、染色反应、培养性状和生理生化反应进行系统研究。结果上述3个菌株均属于芽胞杆菌属(Bacillus),1号菌株为巨大芽胞杆菌(Bacillus megaterium),2号菌株为枯草芽胞杆菌(Bacillus subtilis),3号菌株为地衣芽胞杆菌(Bacillus licheniformis)。结论小碎斑鱼蛉幼虫肠道环境中的不同细菌,其数量存在明显差异。     

黄粉虫不同虫态肠道细菌分离及鉴定

目的研究黄粉虫肠道细菌的类群与其取食的关系,为进一步开发利用提供理论依据。方法从人工饲养的黄粉虫幼虫、蛹和成虫肠道环境中分离纯化获得9个细菌菌株,对其菌体形态、染色反应、培养性状、生理生化反应进行系统研究。结果研究结果表明,上述9个细菌菌株分别属于放线杆菌属（Actunobacillus）、丙酸杆菌属（Propionibacterium）、柠檬酸杆菌属（Citrobacter）、沙雷菌属（Serratia）、芽胞杆菌属（Bacillus）、皮杆菌属（Dermabacter）、短状杆菌属（Brachybacterium）、棍状杆菌属（Clavibacter）和微小杆菌属（Exiguobcacterium）。结论通过对黄粉虫不同虫态即幼虫、蛹和成虫肠道细菌分离,其细菌种类存在一定差别。     

The effects of neurotensin, naloxone and haloperidol on elements of excessive grooming behavior induced by bombesin

The influence of naloxone, haloperidol and neurotensin was investigated on bombesin-induced excessive grooming in rats. All three drugs reduced the amount of bombesin-induced grooming. Haloperidol induced a general reduction in excessive grooming as induced by bombesin, without changing the composition of grooming behavior, whereas naloxone and neurotensin suppressed bombesin-induced grooming and caused a shift in the distribution of grooming elements. The main suppressive effect of these latter drugs appeared to be on the element scratching. From these data it is suggested that bombesin-induced scratching is mainly displayed by activation of opiate receptor systems, whereas the other elements of bombesin-induced excessive grooming are mainly regulated by dopaminergic systems.