Multidimensional Chromatographic and Hyphenated Techniques for Hydrophilic Copolymers, 1

Summary: The chromatographic analysis of hydrophilic copolymers is complicated due to the fact that in most cases aqueous eluents must be used. In aqueous eluents different polar and ionic effects may disturb the selective interactions between the macromolecules and the stationary phase making it impossible to separate such copolymers with regard to chemical composition. Therefore, 2D chromatography combining a separation according to composition with a separation according to molar mass has been applied mostly to polymers that are soluble in organic solvents. The present contribution describes experimental approaches to analyze such hydrophilic copolymers by 2D‐chromatography. For a model polymer system resulting from the copolymerization of methacrylic acid and a poly(ethylene glycol) macromonomer, it is shown that different analytical techniques including SEC, LC‐CC, MALDI‐TOF MS and 2D chromatography can be used to analyze the different parameters of molecular heterogeneity of such copolymers.

2D separation of poly(MPEG‐MM 2), 1^st dimension: LC‐CC, 2^nd dimension: SEC.     

Excitatory effect of dehydroepiandrosterone, its sulphate ester and pregnenolone sulphate, applied by iontophoresis and pressure, on single neurones in the septo-preoptic area of the guinea pig

When dehydroepiandrosterone (D), its ester sulphate (DS) and pregnenolone sulphate (PS) were applied iontophoretically or by pressure to neurones in the septo-preoptic area, an excitatory effect was observed. DS and PS, applied on the same neurone, always produced a similar effect. When DS and D were tested, some neurones were excited by both steroids whereas others responded to DS but were unaffected by D. DS-, PS- and D-induced responses displayed a short latency in onset and offset, suggesting an action at the membrane level as for other steroids.     

Biochemical characterization of I-Ak proteins from mutant antigen-presenting B-cell--B-lymphoma hybridomas

Chemically induced mutants of an I-Ak,d expressing antigen-presenting B-cell--B-lymphoma hybridoma have recently been generated by immunoselection in vitro and were found to possess alterations in some of their serologically and functionally defined I-Ak region dependent functions. In order to identify at the structural level the origin of the differences in serological and functional properties of these mutants, I-Ak molecules from several of these mutant hybridomas were compared biochemically to wild-type I-Ak polypeptides by two-dimensional gel electrophoresis and high-pressure liquid chromatographic tryptic peptide analyses. Two-dimensional gel electrophoresis indicated that no major structural alterations, resulting in changes in mol. wt or charge, had occurred in the Ak alpha or Ak beta polypeptides from the mutant cells. Likewise, Ak alpha peptide maps of the mutants were indistinguishable from the normal Ak alpha peptide maps. However, two of the three mutants studied did exhibit one additional peptide in their Ak beta peptide maps. These results suggest that the major deficiencies in T-cell-activating functions of these mutants are a result of a limited alteration in the Ak beta polypeptide primary structure.     

Distribution of galanin immunoreactivity in the central nervous system and the responses of galanin-containing neuronal pathways to injury

Radioimmunoassay and immunocytochemistry were used to study the distribution of galanin, a novel 29 amino acid porcine intestinal peptide, in the central nervous system of the rat and pig. The pattern of distribution was similar in the two species, with the highest concentrations of galanin-like immunoreactivity found in the neurohypophysis, hypothalamus and sacral spinal cord. Immunocytochemical studies of these regions localized galanin-like immunoreactivity to cell bodies in the paraventricular and supraoptic nuclei of the hypothalamus, to fibres in the pars nervosa and to numerous cell bodies and fibres in the dorsal horn of the spinal cord. On both gel and high pressure liquid chromatography, galanin-like immunoreactivity in rat and pig nervous tissue eluted as a single peak in a position similar to purified procine intestinal galanin standard. Surgical and pharmacological manipulations in the rat suggest the presence of galanin in afferent fibres. An increase of galanin-like immunoreactivity was observed in the sacral spinal cord of the rat following thoracic spinal cord transection. Thus galanin-like immunoreactivity in the brain is mainly localized in the hypothalamopituitary region. The decrease of galanin-like immunoreactivity in the dorsal horn of the spinal cord, following dorsal rhizotomy and pre-treatment of rats with capsaicin, indicates that many of the fibres, which are of small diameter, may well be derived from spinal sensory neurones.     

Recognition of a novel naturally processed, A2 restricted, HCV-NS4 epitope triggers IFN-gamma release in absence of detectable cytopathicity

Using short term CTL lines derived from HLA A2/K^b transgenic mice and IFN-gamma release assays we demonstrate that the NS4.1769 epitope, is generated from natural processing of the NS4 antigen, and presented in the context of the A2/K^b molecules. Interestingly, T cell recognition of the naturally processed form of the NS4.1769 epitope was associated with significant IFN-gamma release, but no direct cytolytic activity. Epitopes of this phenotype might be of interest, in terms of therapy of chronic HCV infection by associating the benefit of localized lymphokine release with low or absent direct cytopathicity.     

Assessment of the role of "enkephalinase" in cholecystokinin inactivation

Cholecystokinin octapeptide and the C-terminal tetrapeptide are hydrolysed by a highly purified preparation of "enkephalinase" (EC 3.4.24.11). In both cases the Asp-PheNH2 bond is hydrolysed and the Gly4-Trp5 bond of the octapeptide is also cleaved, though more slowly. Evaluated from the appearance of Phe-NH2, the Km for the hydrolysis of the octapeptide by the purified peptidase is 57 microM and that for the tetrapeptide 65 microM. The apparent affinities of these peptides for the enzyme in striatal membranes are similar. The importance of this hydrolysis in the inactivation of endogenous cholecystokinin was assessed by studying the fate of cholecystokinin immunoreactivity released from slices of rat cerebral cortex and striatum by depolarization with potassium. In the absence of any peptidase inhibitor only 16% of the peptide released from the tissue was recovered in immunoreactive form in the medium, indicating that endogenous cholecystokinin octapeptide is, like other neuropeptides, rapidly and extensively hydrolysed following release. Selective inhibition of "enkephalinase" by Thiorphan (DL-3-mercapto-2-benzylpropanoyl glycine) did not significantly alter the recovery from slices of cerebral cortex and had only a very slight effect in the case of striatal slices. This suggests that, while cholecystokinin octapeptide is a substrate for "enkephalinase", this enzyme plays a less important (if any) role in the inactivation of endogenous cholecystokinin than for the opioid peptides.     

Regional distribution of the 5-HT innervation in the brain of normal and lurcher mice as revealed by [H]citalopram quantitative autoradiography

The neurological cerebellar mutant lurcher is characterized by a primary degeneration of Purkinje cells as well as a retrograde secondary partial degeneration of cerebellar granule cells and inferior olivary neurons. Since serotonin (5-HT) has been implicated in the modulation of excitatory amino acid systems of the cerebellum, the 5-HT innervation of the normal and lurcher mice was examined by quantifying uptake sites using [³H]citalopram autoradiography, and by biochemical assays of the indoles 5-HT, 5-hydroxy- -tryptophan and 5-hydroxyindole-3-acetic acid using high-performance liquid chromatography. Comparable results were found between [³H]citalopram binding and 5-HT tissue concentrations in different brain regions. The highest [³H]citalopram labelling was observed in defined structures of the mesencephalic and upper pontine regions, in limbic structures, in hypothalamus and in discrete thalamic divisions, while the lowest labelling of uptake sites was documented in cerebellum and brainstem reticular formation. In lurcher mutants, the histology confirmed cell degeneration and the reduction in width, leading to 65%, 45% and 25% atrophies of total cerebellum, deep nuclei and inferior olivary nucleus, respectively. The [³H]citalopram labelling corrected for surface loss was 45% and 20% higher in cerebellar deep nuclei and red nucleus, respectively, but remained unchanged in the cerebellar cortex and inferior olivary nucleus. Moreover, higher labelling was found in nucleus raphe dorsalis, ventral tegmental area, inferior colliculus, locus coeruleus, pontine central grey and anterior thalamic nuclei, areas known to be part of cerebellar afferent and efferent systems. The present results indicate that in such pathological conditions as described for the lurcher mutant, the 5-HT system may modulate motor function not only at the level of the cerebellum, but also in other forebrain structures functionally related to the motor system.     

Substance P and somatostatin metabolism in sympathetic and special sensory ganglia in vitro

Mechanisms regulating the content of the putative peptide transmitters, substance P and somatostatin, were examined in several neuronal populations in culture. Substance P levels increased more than 25-fold within 48 h in sympathetic neurons in the explanted rat superior cervical ganglion, and remained elevated for 4 weeks. Identity of the peptide was authenticated by combined high pressure liquid chromatography-radioimmunoassay. Veratridine prevented the increase of substance P in vitro, and tetrodotoxin blocked the veratridine effect, suggesting that sodium ion influx and membrane depolarization prevent peptide elevation. Veratridine (or potassium)-induced membrane depolarization released substance P into the culture medium through a calcium-dependent process. Consequently, at least some veratridine effects are attributable to release and subsequent depletion of ganglion peptide. However, the inhibitory effects of veratridine were far greater than could be accounted for by the quantity of peptide released, suggesting a separate influence on net synthesis (synthesis less catabolism) of substance P. Viewed in conjunction with previous in vivo studies, our observations suggest that trans-synaptic impulses, through the mediation of postsynaptic sodium flux, release substance P from sympathetic neurons and also regulate intracellular peptide metabolism. To determine whether the processes regulating substance P in sympathetic neurons reflect generalized mechanisms, a different peptide, somatostatin, was examined in sympathetic neurons; moreover, substance P was examined in a different neuronal population, special sensory neurons in the nodose ganglion. Substance P levels increased significantly in both sympathetic and sensory neurons after explantation, and somatostatin levels increased in sympathetic neurons. In each instance, the increase was dependent upon the presence of the calcium ions. Moreover, these increases were all prevented by veratridine, in a tetrodotoxin-sensitive manner. Our observations suggest that common regulatory mechanisms govern peptide transmitter metabolism in diverse neuronal populations.     

Properties of the omega-hydroxylation system of docosahexaenoic or arachidonic acid in brain or liver homogenate of rat

The homogenate of a brain or liver obtained from a 1–55-day-old rat was incubated with NADPH and docosahexaenoic or arachidonic acid as the substrate. ω-Hydroxydocosahexaenoic or ω-hydroxyeicosatetraenoic acid from an incubation mixture of the homogenate was detected on a selected-ion monitoring chromatogram of reversed phase-HPLC-thermospray-mass spectrometry. ω-Hydroxylation activity in the brain homogenate considerably increased with growth up to 55 days. Activity in the liver homogenate decreased much with growth up to 55 days. ω-Hydroxylation activity in homogenates of rat brain gray matter, white matter, medula oblongata and cerebellum was much the same. ω-Hydroxylation activity of docosahexaenoic acid in rat brain homogenate was maximal at pH 7.5–8.0 in 50 mM Tris-HCL buffer and was inhibited by CO gas, metyrapone, ADP-Fe³⁺, heat treatment at 100°C for 5 min and without NADPH. Based on these results, it is suggested that ω-hydroxylation activity is associated with cytochrome P-450 without NADPH-ADP-Fe³⁺-dependent lipid peroxidation, and the ω-hydroxylation system may be a metabolic pathway of the fatty acids in adult rat brain or neonatal rat liver. Since ω-hydroxyeicosatetraenoic acid produces relaxation of artery, it is suggested that blood flow changes in rat brain or liver with growth are caused by ω-hydroxylation activity changes in these organs with growth.     

丹酰氯柱前衍生人尿液多胺高效液相色谱(HPLC)快速测定

建立了丹酰氯柱前衍生HPLC法测定人尿中多胺含量的方法。以己二胺为内标 ,uBondapak -C18(2 5 0× 4.6mm ,10um)为固定相 ,甲醇和水为流动相 ,梯度洗脱 ,柱温 5 0℃ ,流速 1ml/min ,荧光检测器测得腐胺 (PUT)、精脒 (SPD)和精胺(SP)三者回收率为PUT 97%、SPD 98%、SP 10 3% ,回归方程线性良好 ( 均大于 0 99) ,分析时间约 9min。该法简洁 ,快速 ,灵敏度高 ,重现性好 ,可有效分析人尿及其他生物样品中的多胺含量