A protocol for the purification of bovine serum albumin free of deoxyribonuclease activity

Bovine serum albumin, free of deoxyribonuclease activity, was obtained in our laboratory using ion-exchange chromatography followed by acetylation. Chromatography on four different resins (DEAE-52, P-11, hydroxylapatite and Q Sepharose fast-flow) was examined. Fractions from Q Sepharose chromatography, eluted with a linear gradient 0-1.0 M NaCl and subsequently acetylated, proved to be the most effective method for obtaining deoxyribonuclease-free bovine serum albumin.     

Mechanisms of degradation of 2′-5′ oligoadenylates

We have studied the mechanisms of breakdown of 2'-5' oligoadenylates. We monitored the time-courses of degradation of ppp(A2'p5')nA (dimer to tetramer) and of 5'OH-(A2'p5')nA (dimer to pentamer) in unfractionated L1210 cell extract. The 5' triphosphorylated 2'-5' oligoadenylates are converted by a phosphatase activity. However, 2'-5' oligoadenylates are degraded mainly by phosphodiesterase activity which splits the 2'-5' phosphodiester bond sequentially at the 2' end to yield 5' AMP and one-unit-shorter oligomers. The nonlinear least-squares curve-fitting program CONSAM was used to fit these kinetics and to determine the degradation rate constant of each oligomer. Trimers and tetramers, whether 5' triphosphorylated or not, are degraded at the same rate, whereas 5' triphosphorylated dimer is rapidly hydrolyzed and 5'-OH dimer is the most stable oligomer. The interaction between degradation enzymes and the substrate strongly depends on the presence of a 5' phosphate group in the vicinity of the phosphodiester bond to be hydrolyzed; indeed, when this 5' phosphate group is present, as in pp/pA2'p5'A/or A2'/p5'A2'p5'A/, affinity is high and maximal velocity is low. Such a degradation pattern can control the concentration of 2'-5' oligoadenylates active on RNAse L either by limiting their synthesis (5' triphosphorylated dimer is the primer necessary for the formation of longer oligomers) and/or by converting them into inhibitory (e.g., monophosphorylated trimer) or inactive (e.g., nonphosphorylated oligomers) molecules.     

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.     

The synergistic lethal interaction of cis-diamminedichloroplatinum and natural nucleosides is related to increased DNA cross-links

Human tumor cells were treated in vitro with combinations of cis- or trans-dichlodiammineplatinum (DDP) and natural nucleosides (thymidine, uridine, cytidine and adenosine). Effects were measured by inhibition of colony-formation (cell survival) and DNA alkaline elution (DNA cross-links). No increments in cell lethality or DNA cross-links were elicited by any combination of trans-DDP and nucleosides. In contrast, every combination of cis-DDP and nucleoside was eminently synergistic with 5- and 10-fold increases in cell lethality over the predicted sum of each agent alone. These increments in cell kill correlated linearly with increases in DNA crosslinks suggesting that the nucleosides interact with cis-DDP to enhance its cytotoxic crosslinking mode of action.     

Cultured human tumour cells may be arrested in all stages of the cycle during stationary phase: demonstration of quiescent cells in G1, S and G2 phase

Six human colon carcinoma cell lines were induced to enter stationary phase of growth by nutrient deprivation and cell crowding. Growth kinetics parameters (cell number, flow cytometric analysis of DNA distribution, and labelling and mitotic indices) were measured sequentially for all lines during the various stages of in vitro growth. Our results demonstrated that a substantial fraction of cells (9-18%) were located in G2 phase when they changed from an exponential to a stationary mode of growth. Moreover, a large number of cells in stationary phase of growth had an S-phase DNA content, as determined by flow cytometry, but failed to incorporate radioactive DNA precursors (up to 15-fold difference). To substantiate these findings, cells in stationary phase of growth were induced to enter exponential growth by re-seeding in fresh medium at a lower density. Subsequently observed changes in DNA-compartment distribution, and in labelling and mitotic indices were those expected from cells that had been arrested at different stages of the cycle during their previous stationary phase. Thus, the non-proliferating quiescent state (Q), traditionally located 'somewhere' in G1 phase, appears to be composed also of cells that can be arrested at other stages of the cycle (Qs and QG2). Although the proportion of such cells is rather small, their contribution to the growth kinetics behaviour of human in vivo tumours will become apparent following 'recruiting' or 'synchronizing' clinical manoeuvres and will prevent the formation of a clear-cut wave of synchronized cells.     

Intensity of photosynthesis and chlorophyll content as related to leaf age inNicotiana Sanderae hort

U r?zně starých list? v listové r??ici 90 a? 110 denních rostlin Nicotiana sanderae hort. byly sledovány rozdály v intensitě ?isté fotosynthesy a v obsahu chlorofylu (a + b). Ke stanovení intensity fotosynthesy bylo pou?ito dvou odli?ných metod, a to váhového stanovení p?ír?stku su?iny podle Barto?e, KubÍna a ?et-lÍka (1960) a gazometrického stanovení infra?erveným analyzátorem CO₂. Nejvy??í intensitu fotosynthesy i nejvy??í obsah chlorofylu (vzhledem k plo?e listové) mají mladé, ale ji? dob?e rozvinuté listy, tj. t?etí a? ?tvrté od vrcholu (prvním listem se rozumí list o plo?e asi 20 cm²). Tyto listy nazýváme ?fotosyntheticky dospělými“. Listy nejmlad?í a zejména pak listy star?í mají intensitu fotosynthesy i obsah chlorofylu ni??í; u nejstar?ích list? je intensita fotosynthesy prakticky nulová. Intensita fotosynthesy i obsah chlorofylu se během vývoje mění: jejich momentální rozdíly u list? v genetické spirále jsou z?ejmě shodné s jejich změnami v ontogenesi listu. Pokles intensity fotosynthesy p?i stárnutí list? je rychlej?í ne? pokles obsahu chlorofylu. P?i ur?itém obsahu chlorofylu (tj. asi 2,25 a? 2,45 mg/dm²) klesá intensita ?isté fotosynthesy k nule. Intensita fotosynthesy je v lineárním vztahu k mno?ství chlorofylu (p?i p?epo?tu na plo?nou jednotku), a to nezávisle na poloze listu v genetické spirále. Obě pou?ité metody ke stanovení intensity fotosynthesy poskytly obdobné výsledky.     

Structure of Major Surface Determinants and DNA Diagnosis of Pneumocystis carinii

Pneumocystis carinii is a pathogen which, causes fatal pneumonia in patients with the acquired immune deficiency syndrome (AIDS). To facilitate the basic study of P. carinii , we have analyzed its major surface proteins by both immunochemical and biochemical methods. The major protein components of both cysts and trophozoites are a group of proteins called "P115" with apparent masses of 105–120 kd. It includes 6 isoelcclric variants. A monoclonal antibody raised against cysts recognizes all 6 variants and reacts with epitopes located in the cell wall indicating that P115 is an immunorcactive surface component. The isoelectric variants contain identical or closely related protein components and they are mannose-rich glycoproteins. The isoelectric variation may be due primarily to differences in glycosylation. The majority of sera from humans with diagnosed pneumocystosis that were tested reacted strongly with the P115 proteins. To develop probes for DNA diagnosis and to facilitate molecular studies, a genomic DNA library of P. carinii has been constructed. Some of these clones were used for DNA hybridization analysis of rat and human lungs.     

镉引起小麦苗逆境乙烯的产生及其和镉的吸收分布的关系

溶液培养小麦幼苗转移至含Cd~(2 )的营养液中,根系乙烯产生较快地增加,约在12h达高峰,然后下降;ACC含量亦呈先升后降的趋势。未和Cd~(2 )溶液直接接触的地上部乙烯亦增加,至36h达高峰,此后急剧下降,而ACG和 MAGC含量持续上升。地上部乙烯的增加,主要是由通过根系运往地上部的镉直接作用的结果,不是根部合成ACG运往地上部后再产生的。电镜观察表明,地上部乙烯产生和ACC含量变化的时间进程,可以与镉进入叶细胞内的部位及其对细胞膜和细胞器的影响相联系。     

固定化虫荧光素酶光纤传感器

固定化虫荧光素酶光纤传感器蔡谨,王顺光,杨歧生,吉鑫松（浙江大学化工系生化教研室,杭州３１００２７;中国科学院上海生物化学研究所,２０００３１）关键词虫荧光素酶,ＡＴＰ,固定化酶,光纤生物传感器ＡＴＰ是生物体内极为重要的能量物质。如何准确快速地定量Ａ...