培养的人脐静脉内皮细胞合成的蛋白聚糖

以[~(35)S]-Na_2SO_4为示踪物,观察培养的人脐静脉内皮细胞(EC)合成及分泌的蛋白聚糖(PG),经DEAE-Sephacel离子交换及Sepharose6B凝胶滤柱层析分析发现细胞层及培养液均含有三种PG单体,即硫酸乙酰肝素蛋白聚糖(HS-PG)、硫酸软骨素蛋白聚糖(CS-PG)及硫酸皮肤素蛋白聚糖(DS-PG)HS-PG又可分为大小两种,前者(HS-PG_L)位于V_o处,后者(HS-PG_s)Kd=0.53(sepharose6B);CS-PG/DS-PG分为三个峰,峰Ⅰ位于V_0处,峰Ⅱ、峰Ⅲ的Kd值分别为0.26及0.52(sepharose6B)。汇合前后细胞层及培养液中各种PG的含量不同。细胞层PG总量汇合前低于汇合后,无论是细胞层还是培养液汇合前HS-PG_L均低于汇合后,HS-PG_L与HS-PG_s比值亦为汇合前低于汇合后,而CS-PG/DS-PG含量则高于汇合后。汇合前后EC合成及分泌PG的差异与文献报道的EC损伤及正常者类似。     

转化细胞及腹水癌细胞对生长调节因子敏感性的研究

 本文~3H-TdR参入细胞DNA为指标研究了EGF等生长调节因子对小鼠腹水癌细胞DNA合成的影响,发现不同癌细胞对EGF等生长因子的敏感性有所差异,考虑到这也许与肿瘤细胞自身特性如恶性度有关。为了进一步探讨恶性度与这一敏感性是否相关,我们观察并比较了C_3H10T1/2CL_8(一种来源于鼠胚的正常成纤维细胞,简称NC_3H_(10)及转化的C_3H_(10)T1/2CL_8(用~3H-TdR转化的上述细胞,简称TC_3H_(10))对EGF等生长因子的敏感性。实验证明,细胞恶性转化后,对EGF的敏感性明显降低,~3H-TdR参入率降至原先的1/4以下。用DBcAMP作用于NC_3H_(10)和TC_3H_(10)均能抑制~3H-TdR参入DNA并可抑制EGF诱导的~3H-TdR参入作用。因此,我们认为,有关物理的致癌因素如放射性同位素,像生物、化学的致癌因素一样,亦能引起其转化细胞对外源性生长调节因子敏感性的改变。     

Role of the N-terminal region of the a chain in β1-bungarotoxin from the venom ofBungarus multicinctus (Taiwan-banded krait)

The N-terminal α-amino groups of β₁-bungarotoxin (β₁-Bgt) fromBungarus multicinctus venom were modified with trinitrobenzene sulfonic acid and the modified derivative was separated by high performance liquid chromatography. The trinitrophenylated (TNP) derivative contained two TNP groups at the α-amino groups of A chain and B chain and showed a marked decrease in enzymatic activity. Methionine residues at positions 6 and 8 of the A chain were oxidized with chloramine T or cleaved with cyanogen bromide to remove the N-terminal octapeptide. Oxidation of methionine residues and removal of the N-terminal octapeptide caused a precipitous decrease in enzymatic activity, whereas antigenicity remained unchanged. The presence of dihexanoyllecithin influenced the interaction between β₁-Bgt and 8-antilinonaphthalene sulfonate (ANS) and revealed that β₁-Bgt consists of two types of ANS-binding sites, one at the substrate binding site of the A chain and the other might be at the B chain. The modified derivatives still retained their affinity for Ca²⁺ and ANS, indicating that the N-terminal region is not involved in Ca²⁺ and substrate binding. A fluorescence study revealed that the α-amino group of the A chain was in the vicinity of substrate binding site and that the TNP α-amino groups were in proximity to Trp-19 of the A chain. In addition, the study showed that the N-terminal region is important for stabilizing the architectural environment of Trp-19. The results, together with the proposal that Trp-19 of the A chain is involved in substrate binding, suggest that the N-terminal region of the A chain plays a crucial role in maintaining a functional active site for β₁-Bgt.     

Molecular cloning of the cDNA for the catalytic subunit of human DNA polymerase delta.

The cDNA of human DNA polymerase delta was cloned. The cDNA had a length of 3.5 kb and encoded a protein of 1107 amino acid residues with a calculated molecular mass of 124 kDa. Northern blot analysis showed that the cDNA hybridized to a mRNA of 3.4 kb. Monoclonal and polyclonal antibodies to the C-terminal 20 residues specifically immunoblotted the human pol delta catalytic polypeptide. A multiple sequence alignment was constructed. This showed that human pol delta is closely related to yeast pol delta and the herpes virus DNA polymerases. The levels of pol delta message were found to be induced concomitantly with DNA pol delta activity and DNA synthesis in serum restimulated proliferating IMR90 cultured cells. The human pol delta gene was localized to chromosome 19 by Southern blotting of EcoRI digested DNA from a panel of rodent/human cell hybrids.     

Exon skipping in human beta-casein.

Earlier amino acid alignments of mature beta-caseins showed that the human protein was shifted in alignment relative to other species, with amino acid deletions in the N-terminal region and others inserted in the C-terminal region. Our alignment, based on cDNA sequences and their translation products, has shown that the amino acid deletions correspond exactly to exon 3 in the other species. Cloning and sequencing of a segment of the human beta-casein gene between exons 2 and 4 revealed the presence of an intact exon 3 sequence in the gene. An interruption of the polypyrimidine tract adjacent to the 5' end of exon 3 sequence may account for the omission of the exon from human beta-casein mRNA.     

PCR amplification of chromosome-specific DNA isolated from flow cytometry-sorted chromosomes.

We have established a method for amplifying and obtaining large quantities of chromosome-specific DNA by linker/adaptor ligation and polymerase chain reaction (PCR). Small quantities of DNA isolated from flow cytometry-sorted chromosomes 17 and 21 were digested with MboI, ligated to a linker/adaptor, and then subjected to 35 cycles of PCR. Using this procedure, 20 micrograms of chromosome-specific DNA can be obtained. Southern blot analysis using several DNA probes previously localized to chromosomes 17 and 21 indicated that these gene sequences were present in the amplified chromosome-specific DNA. A small quantity of the chromosome-specific DNA obtained from the first round of PCR amplification was used to amplify DNA for a second, third, and fourth round of PCR (30 cycles), and specific DNA sequences were still detectable. Fluorescence in situ hybridization using these chromosome-specific DNA probes clearly indicated the hybridization signals to the designated chromosomes. We showed that PCR-amplified chromosome 17-specific DNA can be used to detect nonrandom chromosomal translocation of t(15;17) in acute promyelocytic leukemia by fluorescence in situ hybridization.