Caiman latirostris (broad-snouted caiman) as a sentinel organism for genotoxic monitoring: Basal values determination of micronucleus and comet assay

Caiman latirostris is one of the two crocodilian species that inhabit Argentina. In this country, as a consequence of agricultural frontiers expansion during the last years, many areas of the geographic distribution of the broad snouted caiman overlap with regions of intensive agricultural activity. Contaminants released to the environment may induce genetic alterations in wildlife, which could lead to mutations and/or carcinogenesis. Up to the moment, no studies had been made concerning the possibbility to apply biomarkers of genotoxic evaluation in C. latirostris.The aim of this study was to adapt two widely used genotoxic techniques, the comet assay and the micronucleus test, for their application in C. latirostris and to determine the baseline values in this species, in order to establish its suitability as a sentinel organism for future genotoxic monitoring of environmental pollutants.A total of 41 juvenile caimans of 4 months old (FMO) and 10 months old (TMO) were used. Genotoxic techniques were applied on peripheral blood erythrocytes introducing the necessary modifications required by the material, which are presented here.Our results show that baseline values of DNA damage are quite stable among juvenile caimans (MN: FMO animals 0.87 ± 0.74 and TMO animals 1.04 ± 0.92; DI: FMO animals 103.40 ± 3.36 and TMO animals 120.08 ± 11.33), being independent of the nest of origin, sex and size of the animals and confirm the potential value of both short term tests as accurate screening tools for the evaluation of genotoxic agents in C. latirostris. This is the first reference to the application of genotoxic techniques on C. latirostris and the second in crocodilians.Data provided here will be useful for future studies involving the biomonitoring of natural regions where C. latirostris occurs, employing this species as a sentinel organism for genotoxic assessment of environmental pollutants.     

用氨气敏电极测定L—天门冬酰胺酶的活力

本文建立了用氨气敏电极测定发酵液和细胞悬浮液中Ｌ－天门冬酰胺酶的方法,发现样品加入的一些化学试剂对测定结果有影响,而将样品稀释到一定程度可以消除这些影响。实验结果表明,用氨电极测定的精密度优于用奈斯勒试剂测定的精密度。     

Flow cytometric assay for cytotoxic activity of crude Clostridium perfringens enterotoxin using non-adherent cell FM3A

Abstract The flow cytometric assay method was tested for the cytotoxic activity of Clostridium perfringens enterotoxin (CPE) in culture using mouse mammary carcinoma cell line FM3A stained with propidium iodide (PI). From the results obtained, FM3A cells proved to be susceptible to CPE. A reproducible dose-response curve with FM3A was obtained between crude CPE at 13.9–109 ng/ml and between purified CPE at 40–400 ng/ml, respectively. These findings indicate that non-adherent FM3A is preferable to determine the cytotoxic activity of CPE because it can be used without detachment procedures with trypsinin compared with adherent African monkey kidney cell line (Vero cells). Furthermore, the flow cytometry with non-adherent cell FM3A stained with PI only proved to be a useful method to determine the biological activity of CPE in culture isolates.     

An improved fluorescent substrate for assaying soluble and membrane-associated ADAM family member activities

A fluorescent resonance energy transfer substrate with improved sensitivity for ADAM17, −10, and −9 (where ADAM represents a disintegrin and metalloproteinase) has been designed. The new substrate, Dabcyl-Pro-Arg-Ala-Ala-Ala-Homophe-Thr-Ser-Pro-Lys(FAM)-NH₂, has specificity constants of 6.3 (±0.3) × 10⁴ M⁻¹ s⁻¹ and 2.4 (±0.3) × 10³ M⁻¹ s⁻¹ for ADAM17 and ADAM10, respectively. The substrate is more sensitive than widely used peptides based on the precursor tumor necrosis factor-alpha (TNF-alpha) cleavage site, PEPDAB010 or Dabcyl-Ser-Pro-Leu-Ala-Gln-Ala-Val-Arg-Ser-Ser-Lys(FAM)-NH₂ and Mca-Pro-Leu-Ala-Gln-Ala-Val-Dpa-Arg-Ser-Ser-Arg-NH₂. ADAM9 also processes the new peptide more than 18-fold better than the TNF-alpha-based substrates. The new substrate has a unique selectivity profile because it is processed less efficiently by ADAM8 and MMP1, −2, −3, −8, −9, −12, and −14. This substrate provides a unique tool in which to assess ADAM17, −10, and −9 activities.     

IFA筛选经EMS诱导的沙眼衣原体突变株

目的:用甲基磺酸乙酯(Ethylmethylsulfone,EMS)诱导D型沙眼衣原体突变,利用间接免疫荧光法筛选出突变菌株,为研究不同衣原体基因的功能提供实验依据。方法:将D型沙眼衣原体标准株接种Mc Coy细胞,加入EMS诱导突变,收集存活菌株,利用空斑实验进行衣原体的分离和纯化,并用不同衣原体蛋白单克隆抗体做间接免疫荧光实验筛选突变株。结果:用间接免疫荧光筛选经EMS作用的沙眼衣原体,筛选出三株包涵体形态偏小的菌株(56#、58#、95#),一株圆形包涵体的突变株(61#)和一株D413N表达阴性的突变菌株(83#)。结论:用EMS作为诱导剂诱导D型沙眼衣原体突变,并成功筛选出三种突变株。为寻找衣原体功能基因与衣原体表型之间的联系奠定了实验基础。     

Spectrophotometric Assay of Lipase Activity: A New 4-nitrophenyl Ester of a Dialkylglycerol Suitable as a Chromogenic Substrate of Pseudomonas cepacia Lipase

Evaluation of Pseudomonas cepacia lipase (PCL) activity by a titrimetric method with triacylglycerols (TAG) and synthetic dialkylglycerol esters (DAGE) established the chain length selectivity of the enzyme and this information has been used to design a new chromogenic substrate [1,2-di-O-octyl-sn-glycerol-3-O-(4-nitrophenyl) glutarate] for the determination of the lipolytic activity of PCL.     

Human mesenchymal stem cells are resistant to cytotoxic and genotoxic effects of cisplatin in vitro

Mesenchymal stem cells (MSCs) are known for their important properties involving multilineage differentiation potential., trophic factor secretion and localization along various organs and tissues. On the dark side, MSCs play a distinguished role in tumor microenvironments by differentiating into tumor-associated fibroblasts or supporting tumor growth via distinct mechanisms. Cisplatin (CIS) is a drug widely applied in the treatment of a large number of cancers and is known for its cytotoxic and genotoxic effects, both in vitro and in vivo. Here we assessed the effects of CIS on MSCs and the ovarian cancer cell line OVCAR-3, by MTT and comet assays. Our results demonstrated the resistance of MSCs to cell death and DNA damage induction by CIS, which was not observed when OVCAR-3 cells were exposed to this drug.     

Statistical aspects of design and validation of microtitre-plate-based linear and non-linear parallel in vitro bioassays

Assay validation was performed using four consecutive experiments with the related statistical evaluation. A cell-based assay on microtitre plates measured repeatedly within 1 day and on consecutive days was chosen as the model. The following problems were addressed: (i) choosing an appropriate design on a plate to avoid heterogeneities, (ii) quantification of all sources of variability and (iii) selecting between linear and non-linear parallel line assays. A mixed model was used with the random factors: rows, columns and plates and fixed effect factors with either linear or non-linear parallel line models.