抗咯萘啶的伯氏疟原虫感染红细胞多胺量的测定

目的：了解疟原虫的多胺代谢与咯萘啶（ＰＮＤ）抗药性的关系。方法：感染伯氏疟原虫ＡＮＫＡ株（ＰＳ）和由该株培育的中抗ＰＮＤ品系（ＰＲＡ）及高抗ＰＮＤ品系（ＰＲＢ）的昆明株小鼠于腹腔接种（ｉｐ）后ｄ７取血，经薄层层析后用荧光分光光度法测定正常ＲＢＣ、ＰＳ、ＰＲＡ和ＰＲＢ感染ＲＢＣ的丁二胺（ＰＴＣ）、精脒（ＳＰＤ）和精胺（ＳＰＭ）量。另有感染ＰＳ和ＰＲＢ的小鼠于ｉｐ后ｄ６分别１次灌胃（ｉｇ）ＰＮＤ５ｍｇ／ｋｇ和１０ｍｇ／ｋｇ，ｄ７取血，按上述方法测定给药后感染ＲＢＣ的多胺量，并与不给药组比较。结果：ＰＳ感染ＲＢＣ的多胺量均明显高于未感染疟原虫的正常ＲＢＣ，而感染ＰＲＡ和ＰＲＢ的ＲＢＣ多胺量又显著高于ＰＳ感染ＲＢＣ，且多胺量的增高与抗性程度有关。经ＰＮＤ治疗后ＰＳ感染ＲＢＣ的ＳＰＤ和ＳＰＭ较未治疗组显著下降，而ＰＲＢ感染ＲＢＣ则未见明显变化。结论：伯氏疟原虫对ＰＮＤ的抗药性与其多胺代谢有关。     

Effects of gastrin and difluoromethylornithine on growth of human colon cancer

The effect of difluoromethylornithine (DFMO), a specific inhibitor of ornithine decarboxylase activity, was evaluatedin vivo andin vitro on the growth of a gastrin-sensitive human colon carcinoma (WiDr).In vivo, mice bearing the tumor treated with pentagastrin had larger tumors with higher ornithine decarboxylase activity and polyamine content (P<0.05) than mice not treated with pentagastrin. Difluoromethylornithine treatment significantly decreased ornithine decarboxylase in both the pentagastrin-treated and the untreated animals; however, DFMO had no effect on tumor volume, weight, protein, or DNA content. In cell culture, gastrin treatment increased WiDr cell number and [³H]thymidine incorporation in the presence or absence of serum. In serum-free conditions, however, gastrin stimulated cell growth without concomitantly increasing ODC activity. DFMO, on the other hand, decreased both ODC activity and growth. These studies suggest that the trophic effect of gastrin on WiDr human colon cancer is independent of ODC activity. Since gastrin treatment increased ODC activityin vivo, gastrin may interactin vitro with other factors present in serum that can alter ODC activity.Supported by NIH First CA50303 and American College of Physicians Research and Teaching Award to Dr. Smith.     

Autophagy in ageing and ageing-associated diseases

Autophagy is a cell self-digestion process via lysosomes that clears “cellular waste”, including aberrantly modified proteins or protein aggregates and damaged organelles. Therefore, autophagy is considered a protein and organelle quality control mechanism that maintains normal cellular homeostasis. Dysfunctional autophagy has been observed in ageing tissues and several ageing-associated diseases. Lifespan of model organisms such as yeast, worms, flies, and mice can be extended through promoting autophagy, either by genetic manipulations such as over-expression of Sirtuin 1, or by administrations of rapamycin, resveratrol or spermidine. The evidence supports that autophagy may play an important role in delaying ageing or extending lifespan. In this review, we summarize the current knowledge about autophagy and its regulation, outline recent developments ie the genetic and pharmacological manipulations of autophagy that affects the lifespan, and discuss the role of autophagy in the ageing-related diseases.     

Binding of 14C-spermidine to melanin in vivo and in vitro

It has been shown that melanin has the properties of a polyanion and may in vivo and in vitro bind inorganic cations and drugs which are positively charged at physiological pH by a cation-exchange mechanism. In the present study, we explored if the organic aliphatic polycation spermidine would bind to melanin in vivo after administration of ¹⁴C-spermidine to pigmented mice and in vitro at incubations with pigment from beef-eyes. The results showed a high labelling of the pigmented tissues in the mice after the administration of ¹⁴C-spermidine. At long survival intervals, the radioactivity in the melanin was higher than in any other tissue. A strong melanin affinity of ¹⁴C-spermidine was found in vitro. An analysis of the binding by the method of Scale hard showed that the data could be best fitted by the assumption of two classes of binding sites. The in vivo bound material could be displaced by in vitro incubation in solutions containing inorganic cation-chloride salts or HCI and HC1 was also very effective in inhibiting the melanin-binding of ¹⁴C-spermidine in vitro. The results indicate that an electrostatic interaction between spermidine and melanin will occur both in vivo and in vitro.     

Enhanced Uptake of [3H] Spermidine by 9L Rat Brain Tumors After Direct Intratumoral Infusion of Inhibitors of Enzymes of the Polyamine Biosynthetic Pathway

We have been exploring the feasibility of delivering ionizing radiation to brain tumor cells by using tritium labeled polyamines. Polyamines are taken up preferentially by dividing cells and form noncovalent bonds with DNA. Their uptake can be enhanced by drugs which deplete endogenous polyamines. To test this in vivo, 9L cells were implanted in the striatal region of the brain in male Fisher 344 rats. Osmotic pumps containing trace amounts of [³H] spermidine or [³H] putrescine with either difluoromethylornithine or combinations of 3 inhibitors of enzymes of the polyamine biosynthetic pathway were implanted subcutaneously and were connected to intratumoral cannulas. After 14–16 days the brains were removed and sliced in the coronal plane. The diameters of the tumors were measured and tumor tissue was dissected from each slice, weighed and lysed for scintillation counting. It was found that difluoromethylornithine enhanced the uptake of [³H] putrescine while a combination of inhibitors of enzymes of the polyamine biosynthetic pathway enhanced the uptake of [³H] putrescine and [³H] spermidine producing a localized region of radioactivity in the 9L tumor. It is estimated that if the [³H] polyamines were at higher specific activity (commercially available), instead of the trace dose given here, the [³H] polyamine uptake would be sufficient to kill 9L tumor cells within a 2 to 3 week period.     

Early increases in transglutaminase activity and polyamine levels in a Mallory-Denk body mouse model

Rodents treated with 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC) are a model of two hepatic toxic manifestations: porphyria and the appearance of hepatic cytoplasmic protein aggregates (Mallory-Denk Bodies, MDBs). MDBs are induced after long-term DDC feeding, consist primarily of keratins 8 and 18, and contain glutamine-lysine cross-links generated by transglutaminases (TGs). TGs are Ca²⁺-dependent enzymes which catalyze the formation of covalent bonds between proteins and between proteins and polyamines. The aim of the current study was to investigate the time-course of TG hepatic activity in CF1 male mice either acutely or chronically treated with DDC and to correlate this activity with polyamine and porphyrin levels. On day 3 of the treatment, statistically significant increases in TG activity (75%), porphyrin content (6740%) and spermidine levels (73%) were observed. Although not statistically significant, at this time point putrescine levels showed an increase of 52%. The highest TG activity was observed on day 30 (522%), while porphyrin levels were still gradually increasing by day 45 (37,000%). From day 7 of the treatment and until the end of the experiment, putrescine levels remained increased (781%). Spermine levels were not affected by the treatment. The DDC-induced increases in putrescine and spermidine levels herein reported seem to be an early event contributing to the stimulation of liver TG activity, and thus to the promotion of cross-linking reactions between keratin proteins. This in turn would contribute to the formation of protein aggregates, which would lead to the appearance of MDBs. Due to the pro-oxidant and antioxidant properties of polyamines, it is possible to speculate that putrescine and spermidine may also participate at several levels in the oxidative stress processes associated with MDB formation.     

Analysis of erythrocytic polyamines by automated reverse-phase liquid chromatography

A sensitive and reproducible method for quantitation of polyamines in erythrocytes by high-performance liquid chromatography (HPLC) is described. Spermidine and spermine are first converted to fluorescent dansyl derivatives and are then separated in less than 15 minutes on a C18 reverse-phase cartridge using methanol in water mobile phase. Sensitivity of the method is 20 pmol with recoveries that average 96% and 95% for spermidine and spermine, respectively. Precision study revealed an average mean coefficient of variation of 5.42 ± 2.95 (mean ± S.D.) from four levels of the polyamines (8.2 to 354.9 nmol/g hemoglobin [Hgb]). The normal ranges for 30 apparently healthy men and women, aged 20–60 years, were 2.9–33.7 nmol/g Hgb (± 2 S.D. from mean) for spermidine and 0-20.9 nmol/g Hgb for spermine. The quantity of putrescine was negligible. Feasibility of this method was evaluated in serial specimens from an advanced-stage patient with prostate cancer who was receiving multiple-modality therapy. Results revealed that this HPLC method can be used in quantifying circulating polyamines in clinical specimens.     

The effects of buffer cations on interactions between mammalian copper-containing amine oxidases and their substrates

Summary We and others have observed that substrates for copper-containing amine oxidases cause substrate inhibition at high concentrations. Through use of a novel “pseudoquantitative” rapid equilibrium approach, kinetic analyses with human and bovine enzymes indicate that these effects are consistent with substrates binding to oxidised and reduced enzyme forms. Small cations compete with binding of substrates to oxidised and reduced enzyme, influencing both substrate turnover and substrate inhibition patterns. Cations reduce affinity of the resting bovine enzyme for spermidine, but not benzylamine, indicating that the predominant effect of cations on substrate oxidation results from binding to an anionic site outside the active site. However, binding of cations to the active site of the reduced form of both enzymes attenuates substrate inhibition with both spermidine and benzylamine. Our observations have significant practical implications for researchers assaying kinetic behaviour of these enzymes, and particularly those developing novel inhibitors of human copper-containing amine oxidases.     

The mechanisms by which polyamines accelerate tumor spread

Background

The pathogenic role of beta-HPVs in non melanoma skin cancer (NMSC), is not still completely understood, and literature data indicate that they might be at least cofactors in the development of certain cutaneous squamous cell carcinomas. However, only few reports contain data on basal cell carcinoma (BCC). The HPVs interact with many cellular proteins altering their function or the expression levels, like the p16^INK4a and Akt. Our study aimed to determine the presence of different beta -HPV types and the expression of p16^INK4a and Akt in BCC, the commonest NMSC, in the normal appearing perilesional skin and in forehead swab of 37 immunocompetent patients.

Methods

The expression of p16^INK4a and Akt, by immunohistochemistry, and the HPV DNA, by nested PCR, were investigated in each sample.

Results

No correspondence of HPV types between BCC and swab samples was found, whereas a correspondence between perilesional skin and BCC was ascertained in the 16,7% of the patients. In BCC, 16 different types of beta HPV were found and the most frequent types were HPV107 (15,4%), HPV100 (11,5%) and HPV15 (11,5%) all belonging to the beta HPV species 2. Immunohistochemistry detected significant p16^INK4a expression in almost all tumor samples (94,3%) with the highest percentages (> 30%) of positive cells detected in 8 cases. A statistically significant (p = 0,012) increase of beta HPV presence was detected in p16^INK4a strongly positive samples, in particular of species 2. pAkt expression was detected in all tumor samples with only 2 cases showing rare positive cells, whereas Akt2 expression was found in 14 out of 35 BCC (40%); in particular in HPV positive samples over-expressing p16^INK4a.

Conclusions

Our data show that p16^INK4a and pAkt are over-expressed in BCC and that the high expression of p16^INK4a and of Akt2 isoform is often associated with the presence of beta-HPV species 2 (i.e. HPV 15). The association of these viruses with the up-regulation of p16^INK4a and Akt/PI3K pathway suggests that in a subtype of BCC these viruses may exert a role in the carcinogenesis or in other, still undefined, biological property of these tumors. If this particular type of BCC reflects a different biology it will remain undisclosed until further studies on a larger number of samples will be performed.     

亚精胺对盐胁迫下紫苏种子萌发和幼苗生理特性的影响

目的 通过对紫苏种子萌发和幼苗生理特性的研究,寻找提高紫苏种子及幼苗在盐胁迫条件下的抗盐能力的方法.方法 测定不同浓度亚精胺(Spd)处理后的盐胁迫下紫苏种子的发芽势(Gv)、发芽率(Gr)和发芽指数(Gi),并对紫苏幼苗叶片的相对含水量、丙二醛(MDA)和超氧化物歧化酶(SOD)、过氧化物酶(POD)和过氧化氢酶(CAT)的活性进行了测定.结果 盐胁迫下的紫苏种子萌发受到显著抑制,但是经过不同浓度的Spd处理后,萌发指标均有所升高,其中经过0.25 mmol/L Spd处理,各项指标均达到最大,Gv为59.7%,Gr为69.4%,Gi为12.8.Spd处理减缓了盐胁迫下紫苏叶片的相对含水量和总黄酮降低的趋势,并且降低了叶片MDA的量.Spd处理后SOD、POD和CAT3种酶的活性均有所增加,但在Spd为0.25 mmol/L时达到最大值,分别为72.4 U/g、8.7 U/mg、8.8 U/mg.结论 0.25 mmol/L的Spd能够有效地减缓盐胁迫对紫苏种子及幼苗产生的伤害,提高了种子及幼苗的抗盐能力.