The use of micropreparative electrophoresis of protein/peptide isolations for primary structure determinations

High-performance electrophoresis chromatography (HPEC) is a recent development that features continuous-elution gel electrophoresis for isolating proteins or peptides in range of 1 to 300 microgram quantities. Column gel electrophoresis is conducted under thermostated conditions, and the field voltage can be varied within a run with a programmable power supply. Applications of this apparatus in protein purification are presented to demonstrate the utility of the (Model 230A) HPEC. These examples include on-line detection with direct analyte recovery of highly purified sample, which mimics high-performance liquid chromatography, for subsequent structure-function characterization. A method to remove salts from sodium dodecyl sulfate electrophoresed samples for subsequent sequencing or amino acid analysis is described. This desalting procedure recovers from 90%-95% of the sample and employs a low molecular weight cut-off membrane during sample centrifugation onto a polyvinylidene difluoride membrane. Subsequent washings are performed to efficiently remove salts, free amino acids and detergents that are known to interfere with sequence analysis. Sequence information such as initial recovery, repetitive yields and chromatogram comparisons are presented to demonstrate the utility of this procedure when used following isolation of sample with HPEC.     

用免疫荧光技术研究细胞周期中核基质的分布变化

本文利用含有抗核基质自发抗体的硬皮人血清,以小鼠艾氏腹水癌细胞为材料,用间接免疫荧光染色的方法,追踪了对应核基质抗原在细胞周期中分布的变化。结果显示,在末期和间期之间存在一个核基质抗原从细胞质向细胞核内转移的过程。由于这一过程是通过核膜进行的,从而提示核基质结构可能有解聚和再聚合的行为。用酶化学结合间接免疫荧光染色的方法,初步研究了抗原的化学性质。染色形态的比较研究显示所用血清中可能含有不同于以前发现的、抗新的核基质抗原的自发抗体。     

白腹锦鸡机体营养成分的分析与探讨

本文报道笼养和野生白腹锦鸡机体营养成分及其差异。分析表明,笼养的比野生种营养成分含量高的有:腿肌蛋白质高11％,胸肌、腿肌、全血的氨基酸分别高2.64％,1.39％和4.68％,胸肌、腿肌和肝脏的碳水化合物分别高0.076％、0.092％和3.962％,胸肌和腿肌的维生素A分别高188.63和84.09 I.U.,胸肌和腿肌的维生素D分别高47.2和12.8 I.U.。但是胸肌蛋白质含量笼养的比野生的低26％。     

Identification of three novel missense PKU mutations among Chinese.

Three novel missense mutations have been identified in the phenylalanine hydroxylase (PAH) genes of Chinese individuals afflicted with various degrees of phenylketonuria (PKU). A T-to-C transition was observed in exon 5 of the gene, resulting in the substitution of Phe161 by Ser161. Two substitutions, G-to-T and T-to-G, were observed in exon 7, resulting in the substitution of Gly247 by Val247 and Leu255 by Val255, respectively. Expression analysis demonstrated that these mutant proteins produced between 0 and 15% of normal PAH enzyme activity. Population screening of a Chinese sample population indicates that these mutations are quite rare, together accounting for only about 4% of all PKU alleles among the Chinese. The P161S and G247V mutations were each present on a single PAH RFLP haplotype 4 chromosome in patients form Northern China, while the L255V mutation was present on chromosomes of both haplotypes 18 and 21 in patients from Southern China. These results suggest that the remaining 30% of uncharacterized PKU alleles in the Chinese population may bear a large number of relatively rare PAH mutations.     

Purification and characterization of pea chloroplastic phosphoriboisomerase

Pea (Pisum sativum L.) chloroplastic phosphoriboisomerase (EC 5.3.1.6) can be purified to apparent homogeneity in less than 2 days time with a 53% yield. Important steps in the purification include heat treatment and pseudoaffinity chromatography on Red H-3BN Sepharose. The purified isomerase has a subunit molecular mass of 26.4 kD. The N-terminal sequence has been determined through 34 residues. pH optima are 7.8 (ribose-5-phosphate) and 7.7 (ribulose-5-phosphate); K_m values are 0.9 millimolar (ribose-5-phosphate) and 0.6 millimolar (ribulose-5-phosphate). The enzyme is inhibited by erythrose-4-phosphate, sedoheptulosebisphosphate, glyceraldehyde-3-phosphate, and 3-phosphoglycerate at concentrations close to those found in photosynthesizing chloroplasts. Countercurrent phase partitioning experiments indicate that the pea chloroplastic phosphoriboisomerase interacts physically with phosphoribulokinase.     

Brefeldin A's effects on endosomes, lysosomes, and the TGN suggest a general mechanism for regulating organelle structure and membrane traffic.

Addition of brefeldin A (BFA) to most cells results in both the formation of extensive, uncoated membrane tubules through which Golgi components redistribute into the ER and the failure to transport molecules out of this mixed ER/Golgi system. In this study we provide evidence that suggests BFA's effects are not limited to the Golgi apparatus but are reiterated throughout the central vacuolar system. Addition of BFA to cells resulted in the tubulation of the endosomal system, the trans-Golgi network (TGN), and lysosomes. Tubule formation of these organelles was specific to BFA, shared near identical pharmacologic characteristics as Golgi tubules and resulted in targeted membrane fusion. Analogous to the mixing of the Golgi with the ER during BFA treatment, the TGN mixed with the recycling endosomal system. This mixed system remained functional with normal cycling between plasma membrane and endosomes, but traffic between endosomes and lysosomes was impaired.     

Negative control by Sandostatin on pancreatic and duodenal growth: a possible implication of insulin-like growth factor I

This study was undertaken to evaluate the effects of Sandostatin, a potent somatostatin analogue, on pancreatic and intestinal growth and plasma and pancreatic levels of insulin-like growth factor I, a known growth factor. Rats weighing 320-330 g, equipped with an intravenous cannula were infused with either bovine serum albumin or Sandostatin at a dose of 5 micrograms kg-1 h-1 for 7 days. Sandostatin caused significant reductions in pancreatic and intestinal weights accompanied by decreases in total DNA, RNA in both organs and total protein in the intestine while total pancreatic enzymes were increased. Plasma cholecystokinin and insulin-like growth factor I were reduced whereas total insulin-like growth factor I pancreatic content was increased. It is suggested that Sandostatin may reduce growth of these two organs by decreasing cholecystokinin and insulin-like growth factor release and their specific effects at the pancreatic and duodenal cellular level.