Predicting development of sustained unresponsiveness to milk oral immunotherapy using epitope-specific antibody binding profiles

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Vasopressin release and firing of supraoptic neurosecretory neurones during drinking in the dehydrated monkey

A close relationship exists between drinking and the release of vasopressin, the two main factors responsible for the maintenance of body water content. Whereas the participation of peripheral factors, such as oropharyngeal stimulation, seems obvious in the metering of fluid intake and in thirst satiation, very little is known about their influence on vasopressin release. In the present experiments, the influence of drinking on vasopressin release was studied using both biochemical and electrophysiological approaches.In one group of monkeys made thirsty by water deprivation, the subsequent drinking of water during a 5–8 min induced: i) a short-term response, consisting of an abrupt fall in plasma vasopressin concentration which was independent of osmolality, occurred at the time of drinking and was partly reversed after the cessation of drinking, and ii) a longer lasting response, consisting of a slow diminution of plasma vasopressin concentration as the intestinal absorption of water progressed. In another group of thirsty monkeys, extracellular recordings were made during drinking from cells which were identified as neurosecretory neurones of the supraoptic nucleus, a number of them being considered vasopressin secreting on the basis of their phasic pattern of firing. Their firing decreased considerably during the periods of water intake and recovered to control levels immediately after-wards.The decrease in vasopressin release at the onset of water intake, the diminution in the firing rate of the neurones, the short latency and the reversibility of these events after cessation of drinking, suggest that a reflex inhibition of vasopressin-secreting neurones occurs which is probably induced by peripheral stimuli and most likely via oropharyngeal or other visceral receptors. It is postulated that this reflex inhibition of vasopressin release may participate in some active manner in the anticipatory mechanisms of thirst satiation.     

Molecular and biological analysis of echovirus 9 strain isolated from a diabetic child

The full-length infectious cDNA clone was constructed and sequenced from the strain DM of echovirus 9, which was recently isolated from a 6-week-old child at the clinical onset of type 1 diabetes. Parallel with the isolate DM, the full-length infectious cDNA clone of the prototype strain echovirus 9 Barty (Barty-INF), was constructed and sequenced. Genetic relationships of the sequenced echo 9 viruses to the other members of the human enterovirus type B species were studied by phylogenetic analyses. Comparison of capsid protein sequences showed that the isolate DM was closely related to both prototype strains: Hill and Barty-INF. The only exception was the inner capsid protein VP4 where serotype specificity was not evident and the isolate DM clustered with the strain Hill and the strain Barty-INF with echovirus 30 Bastianni. Likewise, the nonstructural protein coding region, P2P3, of isolate DM was more similar to strain Hill than to strain Barty-INF. However, like echovirus 9 Barty, the isolate DM contained the RGD-motif in the carboxy terminus of capsid protein VP1. By blocking experiments using an RGD-containing peptide and a polyclonal rabbit antiserum to the alpha(v)beta(3)-integrin, it was shown that this molecule works as a cellular receptor for isolate DM. By using primary human islets, it was shown that the isolate DM is capable of infecting insulin-producing beta-cells like the corresponding prototype strains did. However, only isolate DM was clearly cytolytic for beta-cells. The infectious clones that were made allow further investigations of the molecular features responsible for the diabetogenicity of the isolate DM.     

Age-related changes in adrenergic neuronal function of rabbit vascular smooth muscle

The adrenergic control of vascular smooth muscle was compared in young and adult rabbits using a variety of in vitro techniques. Norepinephrine (NE) content and accumulation of ³H-NE were not different in blood vessels from the two age groups. In contrast, stimulation-evoked release of endogenous NE was reduced by 40–60% in vessels from the aged animals. Functional studies of smooth muscle contractions were carried out using isolated ring segments of the ear artery. There were no differences in the resting force-response relationship between vessels from young and adult rabbits. Maximum contractile responses to nerve stimulation, NE or KCl were not different in vessels from the two age groups, nor was the NE ED₅₀. However, blockade of the neuronal uptake system with desmethylimipramine produced a greater shift in the NE concentration-response curve in vessels from the young animals compared to the shift in vessels from adult animals. This observation reflects a decline in neuronal NE uptake with age. Although maximal contractile responses to transmural nerve stimulation at 16 Hz were unchanged, responses to stimulation at lower frequencies were reduced in vessels from adult rabbits, an effect which was also enhanced when an antagonist of neuronal uptake was present. Thus, there is a decline in function of adrenergic nerves in adult animals, reflected in a decrease in stimulation-evoked NE release and a decrease in norepinephrine uptake revealed by functional studies. These two effects tend to balance each other, so that there is a small decrease in contractile response to adrenergic nerve stimulation which is exacerbated when neuronal uptake mechanisms are blocked.     

Mechanism of histamine release from rat peritoneal mast cells

Summary The present communication endeavours to elucidate the mechanism of histamine release from rat peritoneal mast cells induced by selective histamine liberators.Of the different enzymatic processes involved in secretion the following are considered: ecto-ATPase activity in the mast cell, pro-esterase-esterase conversion during histamine secretion, cyclic AMP and microtubule association/dissociation, phospholipase A₂ and the effect of phospholipid metabolites on secretion, N-methyl transferase and the methylation of phospholipids and the phosphorylation and desphosphorylation of proteins.     

Dihydropyridine inhibition of neuronal calcium current and substance P release

Dihydropyridine (DHP) calcium channel antagonists, which inhibit the slowly inactivating or L-type cardiac calcium (Ca) current, have been shown to be ineffective in blocking⁴⁵Ca influx and Ca-dependent secretion in a number of neuronal preparations. In the studies reported here, however, the antagonist DHP nifedipine inhibited both the L-type Ca current and potassium-evoked substance P (SP) release from embryonic chick dorsal root ganglion (DRG) neurons. These results suggest that, in DRG neurons. Ca entry through L-type channels is critical to the control of secretion. The inhibition of Ca current by nifedipine was both voltage and time-dependent, significant effects being observed only on currents evoked from relatively positive holding potentials maintained for several seconds. As expected from these results, nifedipine failed to inhibit L-type Ca current underlying the brief plateau phase of the action potential generated from the cell's normal resting potential; likewise, no significant effect of the drug was observed on action potential-stimulated SP release evoked by electrical field stimulation. The results of this work are discussed in terms of an assessment of the role of L-type Ca channels in neurosecretion.This work was supported by United States Public Health Service Grant NS16483 (KD) and by a USPHS Postdoctoral Fellowship (SGR)     

Mast cell activation markers for in vitro study

ABSTRACT

Mast cells (MCs) are well known for their role in allergic conditions. This cell can be activated by various types of secretagogues, ranging from a small chemical to a huge protein. Mast cell activation by secretagogues triggers the increase in intracellular calcium (iCa²⁺) concentration, granule trafficking, and exocytosis. Activated mast cells release their intra-granular pre-stored mediator or the newly synthesized mediator in the exocytosis process, in the form of degranulation or secretion. There are at least three types of exocytosis in mast cells, which are suggested to contribute to the release of different mediators, i.e.,, piecemeal, kiss-and-run, and compound exocytosis. The status of mast cells, i.e., activated or resting, is often determined by measuring the concentration of the released mediator such as histamine or β-hexosaminidase. This review summarizes several mast cell components that have been and are generally used as mast cell activation indicator, from the classical histamine and β-hexosaminidase measurement, to eicosanoid and granule trafficking observation. Basic principle of the component determination is also explained with their specified research application and purpose. The information will help to predict the experiment results with a certain study design.     

Controlled Release of the Indomethacin Microencapsulation Based on Layer-by-layer Assembly by Polyelectrolyte Multilayers

Indomethacin has been encapsulated with polyelectrolyte multilayers for controlled release. Gelatin and alginate were alternatively deposited on indomethacin microcrystals. The released amount of indomethacin from coated microcrystals in pH6. 8 phosphate buffer solution （PBS） was measured with a UV spectrophometer. The polyelectrolyte multilayer capsule thickness was proved to control the release rate. The effects of osmotic pressure existed during the release process of indomethacin from microcapsules coated by （gelatin/alginate） 4.     

Poly(ethylene glycol)-containing Hydrogels for Oral Protein Delivery Applications

Novel pH-sensitive hydrogels were developed as suitable candidates for carriers in bioMEMS devices as well as for oral delivery of therapeutic peptides and proteins due to their ability to respond to environmental pH change. Macromonomers containing various PEG molecular weights were synthesized and used to prepare P(MAA-g-EG) hydrogels were by photopolymerization. P(MAA-g-EG) hydrogels showed a drastic change of the equilibrium swelling ratio between pH 2.2 and 7.0. At pH 7.0, hydrogels with PEGMA2000 exhibited higher swelling ratio than hydrogels with PEGMA1000. For both hydrogels with PEGMA1000 and PEGMA2000, the swelling mechanism became more relaxation-controled as the environmental pH changed from 2.2 to 7.0 due to the ionization of the functional groups in polymer networks at high pH. In vitro release studies of insulin were conducted. P(MAA-g-EG) hydrogels exhibited drastic increase of insulin release as the pH of the medium was changed from acidic to basic. Insulin release from P(MAA-g-EG) hydrogels with PEGMA2000 was slower than from hydrogels with PEGMA1000 at both low and high pH. These results were used to design and improve protein release behavior from these carriers.     

Regulation of GABA release by depolarisation-evoked Ca2+ transients at a single hippocampal terminal

We correlated dynamic changes in free cytosolic [Ca²⁺] ([Ca²⁺]_i) within single presynaptic terminals of cultured hippocampal neurones with the postsynaptic GABA-mediated currents. The local changes in [Ca²⁺]_i and evoked inhibitory postsynaptic currents (eIPSCs) were recorded simultaneously using Fura-2 fluorescence and whole-cell patch-clamp respectively. The Ca²⁺ signals and eIPSCs were evoked by direct extracellular electrical stimulation of a single presynaptic terminal by short depolarising pulses. The presynaptic Ca²⁺ transient was graded by varying the amplitude of extracellular stimulating pulses. The probability of the release event, P, estimated for each stimulation strength, reached a maximum (P=1) when the Ca²⁺ signal became maximal and remained at this level at higher stimulation strength, despite the subsequent decrease in the amplitude of the Ca²⁺ transient. A gradual, linear increase in stimulation amplitude (V_stim) resulted in a bell-shaped dependence of the averaged amplitudes of Ca²⁺ signals and corresponding averaged amplitudes of eIPSCs. Analysis of the eIPSC demonstrated that the decrease in both the mean eIPSC amplitude and the mean quantal content of release resulted from a reduction in the probability of multivesicular release, i.e. in the disappearance of failures and in the decrease of individual eIPSC amplitude. The Ca²⁺ signals of similar amplitude resulted in both random and determinate (non-random) neurotransmitter release. We conclude that depolarisation-induced elevation of [Ca²⁺]_i within the terminal is necessary but not sufficient for activation of vesicular release of neurotransmitter.