Identification of two novel amyloid A protein subsets coexisting in an individual patient of AA-amyloidosis

Amyloid A protein (AA), the major fibril protein in AA-amyloidosis, is an N-terminal cleavage product of the precursor protein, serum amyloid A (SAA). Using mass spectrometry and amino-acid sequencing, we identified and characterized two novel AA protein subsets co-deposited as amyloid fibrils in an patient having AA-amyloidosis associated with rheumatoid arthritis. One of the AA proteins corresponded to positions 2–76 (or 75) of SAA2α and the other corresponded to positions 2–76 (or 75) of known SAA1 subsets, except for position 52 or 57, where SAA1α has valine and alanine and SAA1β has alanine and valine in position 52 and 57, respectively, whereas the AA protein had alanine at the both positions. Our findings (1), demonstrate that not only one but two SAA subsets could be deposited together as an AA-amyloid in a single individual and (2), support the existence of a novel SAA1 allotype, i.e., SAA1^52,57Ala.     

Isolation and characterization of porcine somatotropin containing a succinimide residue in place of aspartate129.

Aspartate129 in porcine somatotropin was converted into a cyclic imide residue (succinimide) under acidic solution conditions. Reversed-phase high performance liquid chromatography was utilized to isolate and quantitate this altered species, which accounted for approximately 30% of the total protein. The molecular mass of this modified species was determined by electrospray mass spectrometry to be 18 Da less than normal porcine somatotropin, indicative of a loss of 1 H2O molecule. Tryptic peptide mapping demonstrated that the peptide composed of residues 126-133 was altered in this modified protein. Amino acid analysis, amino acid sequencing, mass spectrometry, and capillary zone electrophoresis were used to demonstrate that aspartate129 in this peptide had been converted into a succinimide residue. Further confirmation that this peptide contained a succinimide was obtained by hydrolyzing the modified peptide at pH 9.0, which yielded both the aspartate and isoaspartate peptides.     

Interfacial thermodynamics of protein adsorption, ion co-adsorption and ion binding in solution. II. Model interpretation of ion exchange in lysozyme chromatography

In this paper we present a model for the ion exchange effects in protein adsorption. The model is applied to chromatography of lysozyme on strong cation exchanger ‘mono S’. The experimental and general thermodynamic aspects have been discussed in Part 1, the preceding paper. The main modelling assumptions are (i) the charge regulation is confined to the small layer of contact between adsorbed protein and exchanger surface, (ii) the contact layer as a whole is electroneutral and (iii) the number of protein acid/base groups and exchanger surface acid groups which participate in the ion exchange is proportional to the area of the contact layer. The model is fitted to the experimental data by adjustment of only two or three parameters. The experimental co-adsorption numbers are very well reproduced. A few conspicuous features emerge: (i) the number of protein acid/base groups and exchanger surface acid groups in the contact layer varies with the medium conditions, such that the number is higher when the interaction between protein and exchanger surface is stronger. (ii) There is indirect evidence for structural alterations in the upper layers of the exchanger surface: the adsorbed protein is probably partly ‘buried’ in the surface.     

Pharmacological and biochemical characteristics of partially purified GABAB receptor

Pharmacological and biochemical characteristics of the partially purified -aminobutyric acid (GABA)_B receptor using baclofen affinity column chromatography have been examined. The Scatchard analysis of [³H]GABA binding to the purified GABA_B receptor showed a linear relationship and the K_D and B_max values were 60 nM and 118 pmol/mg of protein, respectively. Although GTP and Mg²⁺ did not affect on the GABA_B receptor binding, Ca²⁺ significantly increased [³H]GABA binding to the purified GABA_B receptor in a dose-dependent manner and showed its maximum effect at 2 mM. The enhancement of the binding by Ca²⁺ was found to be due to the increase of Bmax by the Scatchard analysis. The treatments with pronase and trypsin significantly decreased the binding of [³H]GABA, but phospholipase A₂ had no significant effect on the binding. In addition, treatment with glycosidases such as glycopeptidase A and -galactosidase significantly decreased the binding of [³H]GABA to the purified GABA_B receptor. These results suggest that purification of the solubilized GABA_B receptor by the affinity column chromatography may result in the functional uncoupling of GABA_B receptor with GTP-binding protein. Furthermore, the present results suggest that cerebral GABA_B receptor may be a glycoprotein and membrane phospholipids susceptible to phospholipase A₂ treatment may not be involved in the exhibition of the binding activity.Special issue dedicated to Dr. Eugene Roberts.     

Pyridine alkaloid distribution in the hoplonemertines

Hoplonemertines possess a family of pyridine compounds affecting the nervous system (Kem, 1985). Anabaseine, the first pyridine to be isolated, stimulates nicotinic receptors. Two other substances, 2,3-bipyridyl and nemertelline (a tetrapyridyl) were isolated from Amphiporus angulatus. In this study samples of 19 species of hoplonemertines were surveyed for the presence of stable pyridines using thin layer chromatography. Pyridines were selectively detected with the Konig reagent.Pyridines were found to be nearly ubiquitous constituents of this taxonomic group. Nevertheless, individual species often differed in the pyridines present as well as the variety of compounds present. A new Konig-reactive pyridine was found in 11 hoplonemertine species. Only Zygonemertes virescens and Amphiporus lactifloreus contained anabasine. Only A. angulatus contained 2,3-bipyridyl and nemertelline. This initial survey suggests that differences in pyridine compositions between hoplonemertines may be a useful taxonomic character.     

低温对大鼠骨骼肌能量代谢的影响——Ⅰ.高能磷酸化合物含量的变化

本实验选择对低温比较敏感的骨骼肌组织作为实验对象。将19只大鼠随机分成3组,其右后肢经冷冻处理使组织温度分别达 5℃、0℃及-5℃,于复温后不同时间测定骨骼肌中四种高能磷酸化合物AMP,ADP、ATP及CP的含量。结果表明,大鼠骨骼肌组织中高能磷酸化合物含量随冷冻时组织最低温度的下降而不同程度的减少,并且具有明显的时相过程。复温后即刻至4小时下降迅速,4～12小时三组值较为接近,其后随时间延长而明显分离。 5℃组恢复得早而且明显,0℃组恢复程度次于 5℃组,-5℃组含量下降最明显而且几乎不恢复。提示:低温使骨骼肌组织能量的产生和贮存过程受到影响。     

Superior resolution of gamma-crystallins from microdissected eye lens by cation-exchange high-performance liquid chromatography

A novel procedure is presented for the rapid quantitative analysis of eye lens gamma-crystallins and beta s-crystallin by ion-exchange high-performance liquid chromatography on Synchropak CM300. At least six different gamma-crystallin gene products can be resolved from the soluble fraction of calf lens extract. This method is applicable to the analysis of microsections from individual lenses, and can be used to rapidly characterize spatial variations in gamma-crystallin composition which occur with aging and cataractogenesis.     

Purification of human plasma retinol-binding protein by hydrophobic interaction chromatography

Human plasma retinol-binding protein has been purified to homogeneity by a simple method that requires an ammonium sulfate fractionation, a hydrophobic interaction chromatography on phenyl-Sepharose, which dissociates the complex between retinol-binding protein and its carrier, transthyretin, and a gel filtration on Sephadex G-50. The yield of pure protein is comparable or higher than that obtained with the more complex procedures previously reported.     

A mechanism for rich-medium inhibition of the repair of daughter-strand gaps in the deoxyribonucleic acid of UV-irradiated Escherichia coli K12 uvrA

Ultraviolet-irradiated Escherichia coli K12 uvrA(B,C) cells show higher survival if plated on minimal growth medium (MM) rather than on rich growth medium (RM). This phenomenon has been referred to as 'minimal medium recovery' (MMR). UV-irradiated (4 J/m2) uvrA cells showed a similar rate of protein synthesis, whether incubated in MM or RM, however, they showed a severe depression in DNA synthesis when incubated in MM that lasted for about 30 min, and the normal rate of DNA synthesis was not reestablished until about 60 min after irradiation. When a sample of these same cells was switched to RM immediately after UV-irradiation, there was only a slight slowing of DNA synthesis, and the normal rate of synthesis was reestablished by 60 min. An additional mmrA mutation or growth retardation by valine blocked both this extra DNA synthesis in RM, and the inhibitory effect of RM on survival. These findings suggest that the absence of a marked delay in DNA synthesis observed in RM may be responsible for the inhibitory effect of RM on the survival of UV-irradiated excision-deficient cells. Two hypotheses, which are not mutually exclusive, are proposed and supported by data to explain why a fast rate of DNA synthesis after UV-irradiation partially inhibits postreplication repair and enhances cell lethality.     

Changes in synthetic activity of cell walls during the cell cycle in a synchronous culture of Catharanthus roseus

The incorporation rates of [¹⁴C] glucose into various fractions of the cell walls and into the sugar constituent of each fraction were investigated in a synchronous culture of Catharanthus roseus (L.) G. Don in order to elucidate the synthetic aspects of the cell walls during the cell cycle. Changes in the incorporation of radioactivity were closely correlated with changes in the amount of each cell wall fraction as well as with those in sugar composition as reported previously (S. Amino et al. Physiol. Plant. 60: 326–332, 1984). The specific activity of galactose was higher than that of other sugars throughout the cell cycle, and a temporary increase in the incorporation of radioactivity into all cell wall fractions except cellulose was observed just before the increase in cell numbers. The synthetic activities may play key roles in the regulation of cell wall polysaccharide dynamics during the cell cycle.