Proinsulin C-peptide and its C-terminal pentapeptide: degradation in human serum and Schiff base formation with subsequent CO<Subscript>2</Subscript> incorporation

Processing of human proinsulin C-peptide and its C-terminal pentapeptide in blood serum was studied using reverse-phase HPLC and electrospray mass spectrometry. The results reveal degradation of both peptides, with a longer half-life for intact C-peptide than for the C-terminal pentapeptide. Products from C-peptide degradation were not distinguishable from the peptide background, suggesting endopeptidase degradation of C-peptide. In contrast, a set of products from the C-terminal pentapeptide were identifiable and corresponded to successive losses from the N terminus, showing that the pentapeptide is degraded by aminopeptidase in serum. Consistent with this finding, a slower degradation was found for the N-acetyl-protected pentapeptide. Removal of serum proteins by acetone precipitation produced N-terminally carbamate-modified C-peptide via a Schiff base intermediate (a ketimine with acetone), to which CO(2) was added and acetone removed, generating a cyclic side chain via anhydride formation. The modification was not seen with the pyroglutamate form of C-peptide, with the N-terminally acetylated C-peptide, or with a control peptide having N-terminal Phe, but was found with human C-peptide, its N-terminal tetrapeptide, and a rat C-peptide fragment (all with N-terminal Glu). Hence, the modification appears to require N-terminal Glu, but this is not the only prerequisite since the C-terminal pentapeptide and another control peptide (also starting with Glu) were not modified. A peptide aldimine Schiff base leading to CO(2) incorporation was detected with formaldehyde in NaHCO(3). The observation that C-peptide forms Schiff bases with ketones/aldehydes, enhancing covalent attachment of CO(2), may have biological implications.     

Degradation of pheromone biosynthesis-activating neuropeptide (PBAN) by hemolymph enzymes of the tobacco hornworm,Manduca sexta,and the corn earworm,Helicoverpa zea

The tritium-labeled bis-norleucine analog ofHelicoverpa zea pheromone biosynthesis-activating neuropeptide ([³H]NLPBAN) was incubated in vitro with hemolymph fromManduca sexta orH. zea adult females. The incubations resulted in the formation of several tritium-labeled degradation products. At a [³H]NLPBAN concentration of 0.9 μM the degradation proceeded at a very slow but physiologically plausible rate (2–10 fmol/min/μl hemolymph). The primary [³H]NLPBAN degradation reaction inM. sexta hemolymph was not inhibited by 20 μM leupeptin, 0.1 mM amastatin, 1 mM EDTA, 1 mM EGTA, 1 mM 1,10-phenanthroline, or 2 mM 4-(2-aminoethyl)benzenesulfonyl fluoride; but secondary reactions may have been affected, as some of the inhibitors changed the radio-HPLC profile of the degradation products. It is concluded that hemolymph ofM. sexta andH. zea contains peptidase(s) capable of inactivating circulating PBAN.     

Thermogenic effects of thyrotrophin-releasing hormone and its analogues in the rat

Acute or chronic injection of RX 77,368 (a TRH analogue; 1 mg/kg s.c.) stimulated oxygen consumption (VO2) and brown adipose tissue activity in the rat, and decreased weight gain. Other TRH analogues (CG 3509, RGH 2202) and TRH itself also stimulated VO2. These thermogenic actions are probably mediated centrally by stimulation of sympathetic outflow to brown fat.     

苯酚在氨氮体系中的氧化降解研究

研究了氨氮存在下次氯酸钠处理苯酚模拟废水的氧化特性,探讨了苯酚在氨氯体系中的反应途径。实验结果表明:在含氨氮的苯酚废水中加入次氯酸钠,氨氮将与苯酚发生竞争反应。折点加氯曲线表现为当氯与氨氮质量比由5.35上升到27.67时,氨氮去除率的变化趋势滞后;而余氯量则不断减小,没有折点出现。随着氨氮浓度增加,苯酚的氧化降解受到抑制:一方面,苯酚的去除率不断下降;另一方面,体系中检测到一系列氯酚中间产物,其生成量和种类先增加后减少。HPLC分析结果显示体系中生成的氯酚中间产物至少有2种(2-氯酚和4-氯酚),至多有5种(2-氯酚、4-氯酚、2,6-二氯酚、2,4-二氯酚和2,4,6-三氯酚)。其中,2-氯酚和4-氯酚是导致三卤甲烷产生的最有效前体物质,而2,6-二氯酚、2,4-二氯酚和2,4,6-三氯酚则是生成卤乙酸的高活性物质。实验结果对次氯酸钠处理含氨氮的难生化或有毒有机废水具有一定的参考价值。     

The application of the high-performance ion-exchange chromatography for the analysis of bovine milk proteins

A high-performance ion-exchange chromatographic (HPIEC) method for the analysis of milk proteins is described. The technique provides an alternative to other separation methods. The resolution obtained for both the proteins of the casein group and bovine lactoserum is comparable with that of electrophoretic analysis on polyacrylamide gels and low-pressure chromatography.     

The application of the high-performance ion-exchange chromatography for the analysis of bovine milk proteins

Summary A high-performance ion-exchange chromatographic (HPIEC) method for the analysis of milk proteins is described. The technique provides an alternative to other separation methods. The resolution obtained for both the proteins of the casein group and bovine lactoserum is comparable with that of electrophoretic analysis on polyacrylamide gels and low-pressure chromatography.     

Mathematical model of pituitary thyrotropic function

Summary A nonlinear differential equation is used to develop a mathematical model describing the time course of thyrotropin (TSH) concentration in human plasma after thyroliberin (TRH) stimulation. The application of the model to real data shows that pituitary responsiveness to TRH is highest in euthyroidism, reduced in primary hypothyroidism, and lowest in hyperthyroidism.     

Thermogenic effects of thyrotrophin-releasing hormone and its analogues in the rat

Summary Acute or chronic injection of RX 77368 (a TRH analogue; 1 mg/kg s.c.) stimulated oxygen consumption (VO₂) and brown adipose tissue activity in the rat, and decreased weight gain Other TRH analogues (CG 3509, RGH 2202) and TRH itself also stimulated VO₂. These thermogenic actions are probably mediated centrally by stimulation of sympathetic outflow to brown fat.     

Occurrence and biosynthesis of 11 (R)-hydroxy-eicosatetraenoic acid (11-R-HETE) in the Caribbean soft coralPlexaurella dichotoma

Gorgonian soft corals from the Caribbean Sea are known to contain prostaglandin-like compounds as well as other products of arachidonic acid lipoxygenation, and the formation of the latter has been suggested to represent the first step in prostaglandin biosynthesis. Here we present evidence for the presence of 11-R-hydroxy-5Z, 8Z, 12E, 14Z-eicosatetraenoic acid (11-R-HETE), as well as of the enzyme responsible for its biosynthesis, in the Caribbean gorgonianPlexaurella dichotoma. Lipid extracts fromP. dichotoma were purified by conventional SiO₂ column chromatography followed by reversed phase high-pressure liquid chromatography (HPLC). These yielded a component having chromatographic and spectroscopic properties identical to synthetic 11-HETE. Electron impact mass spectrometric analysis of the acetoxy-, methyl ester derivative of the compound confirmed its identity as 11-HETE, while chiral phase HPLC of the methyl ester derivative showed that the stereochemistry of the alcoholic carbon atom wasR. Enzymatically active homogenates fromP. dichotoma were able to convert both unlabelled and [³H] arachidonic acid into 11-HETE. In vitro biosynthesis of the latter metabolite was also observed with homogenates of the Mediterranean gorgonianParamuricea clavata, another non-prostaglandin-containing soft coral, thus suggesting that 11-R-HETE production is not necessarily accompanied by prostaglandin formation in gorgonian corals.     

Intravenous thyrotropin releasing hormone (TRH) enhances the excitatory actions of acetylcholine (ACh) on rat cortical neurons

Summary I. v. administered TRH (1.25-10 mg/kg) enhanced the excitatory actions of iontophoretically applied ACh on spontaneously active cerebral cortical neurons of pentobarbital-anaesthetized rats. These observations are consistent with the hypothesis of a cholinergic-link in the anti-anaesthetic actions of exogenously administered TRH.     

Basal autophagy is involved in the degradation of the ERAD component EDEM1

Little is known about the fate of machinery proteins of the protein quality control and endoplasmic reticulum(ER)-associated degradation (ERAD). We investigated the degradation of the ERAD component EDEM1, which directs overexpressed misfolded glycoproteins to degradation. Endogenous EDEM1 was studied since EDEM1 overexpression not only resulted in inappropriate occurrence throughout the ER but also caused cytotoxic effects. Proteasome inhibitors had no effect on the clearance of endogenous EDEM1 in non-starved cells. However, EDEM1 could be detected by immunocytochemistry in autophagosomes and biochemically in LC3 immuno-purified autophagosomes. Furthermore, influencing the lysosome-autophagy pathway by vinblastine or pepstatin A/E64d and inhibiting autophagosome formation by 3-methyladenine or ATGs short interfering RNA knockdown stabilized EDEM1. Autophagic degradation involved removal of cytosolic Triton X-100-insoluble deglycosylated EDEM1, but not of EDEM1-containing ER cisternae. Our studies demonstrate that endogenous EDEM1 in cells not stressed by the expression of a transgenic misfolded protein reaches the cytosol and is degraded by basal autophagy. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users. Received 15 January 2009; received after revision 16 February 2009; accepted 17 February 2009 V. Le Fourn, K. Gaplovska-Kysela: These authors equally contributed to this work.     

Zur Bindung von Biliverdin an das Protein im Crenilabrus-Blau

Summary The blue biliprotein from the fins of the Mediterranean fishCrenilabrus pavo C.V. was analysed with respect to linkages between colouring matter (biliverdin IX) and apoprotein. It was shown by chromic oxidation under various conditions and determination of degradation products that one of the outer and one of the inner rings of biliverdin are free. The remaining inner ring is bound to the apoprotein presumably by an ester bond, while the lability of the linkage between one outer ring and apoprotein corresponds to a N-acyl-bond.     

Peptide-stimulated release of prolactin from the fowl anterior pituitary gland

Summary Anterior pituitary glands from broiler fowl were preincubated for 24 h in either medium 199 only or medium containing estradiol 17, following which they were incubated in medium containing thyrotrophin releasing hormone (TRH), vasoactive intestinal polypeptide (VIP) or substance P (SP), alone or with the dopamine agonist, apomorphine. Estradiol priming stimulated release of prolactin and enhanced apomorphine-inhibition of prolactin release. TRH stimulated prolactin release, an effect reversed by apomorphine, and priming with estradiol potentiated both effects. VIP stimulated prolactin to a lesser degree and again this was inhibited by apomorphine and potentiated by estradiol. SP had little effect on the nonsteroid-primed pituitary, but stimulated release of prolactin after estradiol treatment, though less effectively than TRH or VIP.     

Effects of thyrotrophic-releasing hormone (TRH) on thermoregulation in the rat

Summary At ambient temperatures (T_a) of both 8 and 22°C, intraventricular administration of TRH (10–80 g) produced a dose-dependent hypothermia in rats. The hypothermia was due to both decreased metabolic heat production and cutaneous vasodilatation. In contrast, at 30°C T_a, TRH increased metabolic heat production (due to behavioral excitation) and led to hyperthermia.This work was supported by the grants from the National Science Council of Republic of China and the Pjing-Ling Neurological Foundation (VGH, Taipei, Taiwan).     

On the time course of thyrotropin suppression by high doses of thyroid hormones

Summary Basal and stimulated TSH decreased progressively. Basal TSH was suppressed below the detection limit of 0.4 U/ml after 74 h in 2 of the T₃ and all of the T₄ treated indiduals. At this time, in both groups 3 individuals could be significantly stimulated by TRH (about 5% of the pretreatment stimulation). There was no significant difference in the time course of suppression obtained by T₃ or T₄, though plasma T₃ levels in the T₄ treated group were considerably lower.Supported by the Deutsche ForschungsgemeinschaftThe excellent technical assistance of MissM. Knöpfle, MissU. Neun and Mr.G. Magin is gratefully acknowledged.     

Antagonism by thyrotropin-releasing hormone (TRH) of pentobarbital-induced hypothermia in rats with brain lesions

Summary Anesthesia with a large dose of pentobarbital (55 mg/kg, i.p.) caused a sustained decrease in brain temperature (Tb), which was monitored with a probe placed in the midbrain reticular formation. The administration of TRH to the lateral ventricle antagonized this hypothermia. None of the acute surgeries examined in this paper (adrenal-demedullectomy, septal knife cuts, electrolytic lesions of the hypothalamus and midbrain knife cuts) had any essential effect on this antagonism by TRH. These results suggest that centrally-administered TRH exerts its effect on thermoregulation, at least in part, through brain structure(s) caudal to the midbrain.     

Antagonism by thyrotropin-releasing hormone (TRH) of pentobarbital-induced hypothermia in rats with brain lesions

Anesthesia with a large dose of pentobarbital (55 mg/kg, i.p.) caused a sustained decrease in brain temperature (Tb), which was monitored with a probe placed in the midbrain reticular formation. The administration of TRH to the lateral ventricle antagonized this hypothermia. None of the acute surgeries examined in this paper (adrenal-demedullectomy, septal knife cuts, electrolytic lesions of the hypothalamus and midbrain knife cuts) had any essential effect on this antagonism by TRH. These results suggest that centrally-administered TRH exerts its effect on thermoregulation, at least in part, through brain structure(s) caudal to the midbrain.     

A simplified method for isocratic HPLC analysis of polyamines

A simple technique is described for the separation and analysis of polyamines in tissues and body fluids, utilizing precolumn clean-up on disposable CM-cellulose columns, followed by an automatable HPLC procedure. Complete separation and analysis takes 12-15 min per sample with sensitivity in the pmole range.     

TiO2薄膜的光电催化性能及电化学阻抗谱研究

用直流磁控溅射法在钛网上制备了TiO2薄膜催化剂,采用电化学阻抗谱（EIS）对其阻抗谱特征进行了表征.同时以偶氮酸性红溶液为模型污染物,验证了此工作电极在不同条件下的光电催化活性与阻抗谱之间的关系.研究表明：光电催化实验中TiO2薄膜在同时有紫外光和外加阳极偏压的情况下,有效实现电子-空穴分离,具有最好的催化活性;最佳的外加阳极偏压值为0.3V;其EIS Nyquist图上阻抗环半径也在此时最小,最容易发生反应,与光电催化实验结果一致.     

Gallen und Gallensäuren Papierchromatographische Untersuchungen

Summary In cases of bile-scretion disturbances substitution-therapy can be performed using suitable animal biles equivalent to human bile. Comparative investigations have been made to establish which animal bile is in practice the most suitable for bile substitutiontherapy based on its bile acid components.A new paper partition chromatographic method has been applied for separation and identification of free bile acids. Ascending development was used on Schleicher & Schüll 2043b Mgl paper impregnated with 20 v/v% propylene glycol in chloroform. The xylene-methylethylketone 1:1 solvent system gave good separation. The bile acids can be detected by immersing the chromatograms in 20 w/v% SbCl₃ in chloroform followed by drying and heating for 5–10 min at 100–110°C. The spots show intense reddish-violet or blue fluorescence in filtered UV-light (see Table 1).These investigations have shown that human and ox biles are, in contrast to pig bile, physiologically related biological substances regarding their bile acid components.