Biochemical and autoradiographic studies of TRH receptors in sections of rabbit spinal cord

Receptors for thyrotropin-releasing hormone (pGlu-His-Pro-NH2, TRH) on thaw-mounted sections of rabbit spinal cord have been identified biochemically and visualized by light microscopic autoradiography. Binding of [3H] [3-Me-His2]TRH to 20 microns sections exhibited high apparent affinity and a pharmacological specificity almost identical to that previously demonstrated for spinal TRH receptors in membranes. In autoradiograms, the highest density of TRH receptors appeared in the substantia gelatinosa of the dorsal gray and around the central canal, with intermediate levels in the ventral gray.     

Effects of secretin and gastric inhibitory polypeptide on human pancreatic growth hormone-releasing factor(1-40)-stimulated growth hormone levels in the rat

Synthetic human pancreatic growth hormone-releasing factor containing 40 amino acids ([hpGRF (1-40)]-OH) significantly stimulated plasma growth hormone (GH) levels in both sodium pentobarbital and urethane anesthetized rats. Synthetic secretin, gastric inhibitory polypeptide (GIP), and glucagon significantly decreased plasma GH levels while synthetic vasoactive intestinal peptide (VIP) had no effect. Secretin and GIP also altered the in vivo plasma GH response to [hpGRF(1-40)]-OH. Whether this effect is the result of an interaction at the pituitary level or is due to an extra-pituitary effect of secretin and GIP awaits further study.     

Studies on the complex formed between glucagon and dicaprylphosphatidylcholine

The interaction between glucagon and dicaprylphosphatidylcholine (DCPC) was studied by fluorescence, circular dichroism and calorimetry, as well as by ¹H- and ³¹P-nuclear magnetic resonance. The water-soluble lipid-protein complex was also characterized by gel filtration and ultracentrifugation. The complex appeared to be monodisperse by sedimentation equilibrium measurements, with a molecular weight of (4.55 ± 0.57)·10⁴. This complex contained approximately 7 molecules of glucagon and 35 molecules of phospholipid. Proton-decoupled ³¹P-NMR spectra of the phospholipid in the lipid-protein complex display narrower resonances than those of sonicated vesicles of DCPC, and ¹H-³¹P coupling could be detected in proton coupled spectra. These NMR results, together with gel-filtration results, suggest that glucagon ‘solubilizes’ phospholipid aggregates, forming a lipid-protein complex which is smaller than sonicated preparations of DCPC. ¹H-NMR resonance of both the methionine methyl group (met-27) and the aromatic envelope of glucagon are broadened by the phospolipid, indicating that the C-terminal region and the aromatic residues are involved in the interaction with the phospholipid. Nuclear magnetic resonance titrations of the imidazole ring C(2) and C(4) protons of the histidine residue of glucagon show that DCPC lowers the pK of the imidazole. The alterations caused by the phospholipid in the far and near ultraviolet CD spectra of glucagon reflect, respectively, the increased helix content of the hormone and the fact that the aromatic residues are located in a more structured environment. The phospholipid also alters the fluorescence properties of glucagon, shifting the fluorescence emission maximum of the hormone to shorter wavelength, and enhancing its relative intensity. This suggests that the fluorophore is experiencing a more hydrophobic environment in the presence of the lipid. Binding of glucagon to the phospholipid was analysed by Scatchard plots of the enhancement of fluorescence caused by the phospholipid and showed that the equilibrium binding constants of glucagon to DCPC are (4.4 ± 0.5)·10⁴M^?1 and (7.5±0.5)·10⁴M^?1, at 15°C and 25°C, respectively. The average number of moles of phospholipid bound per mole of glucagon is 4.4±0.6. The isothermal enthalpy of reaction of glucagon with DCPC is ?20.5 kcal/mol of glucagon at 25°C and ?32.5 kcal/mol of glucagon at 15°C. The observed enthalpies can arise from glucagon-induced cyrstallization of the phospholipid, from the non-covalent interactions between the peptide and lipid as well as from the lipid-induced conformational change in the protein. These results demonstrate that, unlike the complexes formed between glucagon and phospholipids which form more stable bilayers, the complex formed between glucagon and DCPC is stable over a wide range of temperatures, including temperatures well above the phase transition.     

A high recovery method for concentrating microgram quantities of protein from large volumes of solution

A method is presented for the recovery of 40-80% of the protein from a 1 microgram/ml solution. The final protein pellet is free of detergent and other ionic compounds and is thus compatible with any denaturing solution. The primary structure of the protein is unaffected by the procedure, making the final pellet an ideal sample for any analytical procedure to determine protein structure.     

The effect of soil water status on critical phosphorus concentrations inStylosanthes hamata cv. Verano

Summary The effect of soil water status on the critical phosphorus concentration (CPC) determined in apices and whole tops ofStylosanthes hamata cv. Verano was investigated in a glasshouse trial. The species was grown with six rates of P and three ranges of soil water potential and was harvested at 10 and 14 weeks after germination. The CPC of both whole tops and apices decliced between the two harvests. At the first harvest the CPC of both whole tops and apices increased as the soil water potential decreased but at the second harvest there was no effect of soil water potential on CPC. It is suggested that at the earlier harvest water stress was delaying physiological development, resulting in a CPC characteristic of chronologically younger tissue, but that by the second harvest the decline in CPC with age had ceased for all water treatments.     

Dose-response comparisons of canine plasma gastroenteropancreatic hormone responses to bombesin and the porcine gastrin-releasing peptide (GRP)

This study compares the potencies of the porcine gastrin-releasing peptide (pGRP) and bombesin, in causing elevations of canine plasma gastroenteropancreatic (GEP) levels. In the dose range 0-600 pmol . kg-1 . h-1, infusion of both peptides resulted in obvious dose-related elevations of plasma levels of gastrin, pancreatic polypeptide, enteroglucagon, immunoreactive pancreatic glucagon, and insulin. In this dose range, no significant difference in potency between the two peptides in elevating plasma levels of the above hormones was observed. The results of this study, demonstrating equimolar potency of pGRP and bombesin, are in contrast to previous studies reporting that pGRP was less potent than bombesin in causing certain bioactivities in the rat following intracranial administration of the two peptides.     

Requirements for antiribosomal activity of pokeweed antiviral protein

It has been known for some time that pokeweed antiviral protein acts by enzymatically inhibiting protein synthesis on eucaryotic ribosome systems. The site of this action is known to be the ribosome itself. In this paper we show that the pokeweed antiviral protein reaction against ribosomes is a strong function of salt concentrations, where 160 mM K⁺ and 3 mM Mg²⁺ retards the reaction, while 20 mM K⁺ and 2 mM Mg²⁺ allows maximum reaction rate. It is also shown, however, that an unidentified protein in the postribosomal supernatant solution, together with ATP, allows the ribosome to be attacked even in the presence of high salt. Kinetic analysis of the antiviral protein reaction has been carried out under both sets of conditions, and reveals that the turnover number for the enzyme is about 300–400 mol/mol per min. in each case. The K_m for ribosomes is 1 μM in the presence of low salt and 0.2 μM at higher salt in the presence of postribosomal supernatant factors plus ATP. The antiviral protein reaction is also shown to be pH dependent and is controlled by a residue with pK_a value of approx. 7.0, apparently a histidine. Stoichiometric reaction of the enzyme with iodoacetamide results in a significant loss of antiribosomal activity.     

Development and specificity of inhibitory terminal arborizations in the central nervous system

This study examined the morphological development of single inhibitory arborizations in the gerbil central auditory brain stem. Using a brain slice preparation, neurons of the medial nucleus of the trapezoid body (MNTB) were filled with horseradish peroxidase (HRP), and their complete arborizations were analyzed along the tonotopic axis of the lateral superior olive (LSO). The projections in neonatal animals displayed well-defined arbors that were ordered appropriately within the LSO. It was evident from the axonal pathways that the MNTB afferents could correct for projection errors after reaching the postsynaptic population. As development progressed, a number of arbors established diffuse or inappropriate projections within the LSO. These immature arborizations were no longer apparent by 18–25 days postnatal. The anatomical specificity of arbors at 12–13 and 18–25 days was quantified by measuring the distance that terminal boutons spread across the frequency axis. There was a significant reduction of this distance in older animals. In addition, there was a significant reduction in the mean number of boutons per arbor between 12–13 days and 18–25 days. The maximum nucleus cross-sectional area continued to increase through 15–16 days, indicating that the refined arbors occupied an even smaller fraction of the postsynaptic structure. Taken together, these observations suggest that central inhibitory arbors form exuberant contacts that must be eliminated during development.     

兔下丘脑室旁核刺激引起的肾交感神经传出活动的抑制

本实验用家兔,氯醛糖及尿酯混合静脉麻醉,制动,人工呼吸,颈部分离出三对神经——迷走、窦及主动脉神经,以备实验中切断。记录股动脉压、肾交感神经传出性放电活动(RSED)及其频率幅度直方图。借助脑立体定向仪刺激下丘脑室旁核,当刺激较强时,在交感神经放电短暂增加之后,可引起血压升高及RSED抑制。这一抑制过程可分为两个时相:血压不变期间的初期抑制时相及与血压升高同时并存的后期抑制时相。实验发现RSED总抑制时程及后期抑制时程均与血压变动具有正相关关系,而初期抑制时程与血压变动无相关关系。切断压力感受性神经前后,虽初期抑制时程的均值无显著差异,但在切断压力感受性神经后,总抑制时程及后期抑制时程的均值大大缩短。当用较弱刺激施于室旁核时,可不引起血压变化,但仍能引起RSED抑制。这个抑制亦可因切断压力感受神经而显著缩短。上述实验结果表明:(1) 在中枢内存在着一个室旁核-肾交感传出系统的抑制机制;(2) 初期抑制来源于中枢性抑制机制,而后期抑制时相主要来源于压力感受性反射,但亦有中枢抑制机制的参与;(3) RSED的中枢抑制可能并不是兴奋后压抑,而是自室旁核至脊髓交感节前神经元的主动性抑制。     

Rhizoplane colonization of pea seedlings by Rhizobium leguminosarum and a deleterious root colonizing Pseudomonas sp. and effects on plant growth

Some pseudomonads produce a toxin that specifically inhibits winter wheat (Triticum aestivum L.) root growth and the growth of several microorganisms. The toxin does not inhibit pea (Pisum sativum) root growth, but the organisms are aggressive root colonizers and their effect on Rhizobium leguminosarum growth, colonization, and nodulation of peas was not known. Peas were grown in Leonard jars in the greenhouse. Pea roots were inoculated with R. leguminosarum, a toxin-producing Pseudomonas sp., both, or neither (control). The Pseudomonas sp. colonized pea roots more rapidly and in greater number than R. leguminosarum after ten days. In the presence of the Pseudomonas sp., the R. leguminosarum population on the rhizoplane was less at ten days. When the roots were inoculated with both R. leguminosarum and Pseudomonas sp., the number of nodules were greater than when R. leguminosarum was inoculated alone, but nodule dry weight and pea shoot biomass were similar to plants inoculated with only R. leguminosarum. Although these results need confirmation with non-sterile soil and field studies, these preliminary results indicate that peas will not be affected by wheat root-inhibitory rhizobacteria.