2, 3, 5-triphenyl tetrazolium chloride (TTC) reduction as exponential growth phase marker for mammalian cells in culture and for myeloma hybridization experiments

Triphenyl tetrazolium chloride in vitro reduction by cells produces a red formazan pellet which can be extracted and measured. We have shown that such reduction is associated with animal cell growth, and particularly with the specific growth rate, so the measurement of Triphenyl tetrazolium chloride reduction is proposed as a physiological marker of the exponential growth of cultured cells. Further application of this technique is shown using this Redox reaction for estimating plasmacytoma fusion potential for hybridoma cell line production.Abbreviations TTC 2,3,5-Triphenyl Tetrazolium Chloride     

Ablation of cereblon attenuates myocardial ischemia–reperfusion injury

Cereblon (CRBN) was originally identified as a target protein for a mild type of mental retardation in humans. However, recent studies showed that CRBN acts as a negative regulator of AMP-activated protein kinase (AMPK) by binding directly to the AMPK catalytic subunit. Because AMPK is implicated in myocardial ischemia–reperfusion (I–R) injury, we reasoned that CRBN might play a role in the pathology of myocardial I–R through regulation of AMPK activity. To test this hypothesis, wild-type (WT) and crbn knockout (KO) mice were subjected to I–R (complete ligation of the coronary artery for 30 min followed by 24 h of reperfusion). We found significantly smaller infarct sizes and less fibrosis in the hearts of KO mice than in those of WT mice. Apoptosis was also significantly reduced in the KO mice compared with that in WT mice, as shown by the reduced numbers of TUNEL-positive cells. In parallel, AMPK activity remained at normal levels in KO mice undergoing I–R, whereas it was significantly reduced in WT mice under the same conditions. In rat neonatal cardiomyocytes, overexpression of CRBN significantly reduced AMPK activity, as demonstrated by reductions in both phosphorylation levels of AMPK and the expression of its downstream target genes. Collectively, these data demonstrate that CRBN plays an important role in myocardial I–R injury through modulation of AMPK activity.     

The use of TTC reduction assay for assessment of Gentiana spp. cell suspension viability after cryopreservation

This study was aimed at improving the 2,3,5-triphenyl-tetrazoliumchloride (TTC) reduction test for initial assessment of cell survival after cryopreservation. Experiments were carried out on three embryogenic cell suspensions of different ages: 9-year-old Gentiana tibetica (King ex Hook. F.), 2-year-old G. kurroo (Royle), and 1-year-old G. cruciata (L.). The suspensions were maintained in MS medium supplemented with 1.0 mg 1⁻¹ 3,6-dichloro-o-anisic acid, 0.1 mg 1⁻¹ naphthaleneacetic acid, 2.0 mg l⁻¹ 6-benzylaminopurine, 80.0 mg 1⁻¹ adenine sulphate and 0.09 M sucrose. Four weeks before freezing, part of the tissue was subcultured to the same medium with sucrose concentrations elevated from 0.09 M (3%sMS) to 0.175 M (6%sMS) or 0.26 M (9%sMS). In freezing treatments without cryoprotection, tissue was plunged directly into liquid nitrogen (LN) or cooled gradually. In freezing treatments with cryoprotection, the cells were pretreated with 1 M sucrose, or with 0.4 M sorbitol + 0.25 M proline or + 0.08 M DMSO, or with vitrification solution (PVS2). Encapsulation was another variant. TTC reduction activity was spectrophotometrically assessed immediately, 1, 3, 5, 24 and 48 h after thawing. Cells without cryoprotection were lethally damaged, but TTC reduction activity in those cells ranged from 6.5% (tissue from 3%sMS) to 73 % (tissue from 9%sMS) directly after thawing. Formazan production was reduced to zero after 24 h. The TTC test showed 50% formazan content immediately after thawing of DMSO-protected G. tibetica tissue, but only 22.47% after 24 h and 2.9% after 48 h. Ultrastructural analysis of those cells showed lethal damage in many of them. For the PVS2 treatment, the formazan content was similar in samples analyzed directly after thawing and 24 h later. Cells treated with PVS2 did not show structural disturbances. Encapsulated cell aggregates of G. cruciata treated with concentrations of sucrose increasing up to 1 M produced 2.6 times more formazan. When applied at least 48 h after thawing, the TTC test can reflect cell viability and can be used to compare the effectiveness of cryoprotectant performance and freezing protocols, but it must be carefully evaluated, with appropriate controls.     

A new case of partial 14q31.3-qter trisomy due to maternal pericentric inversion

Chromosome 14 is often involved in chromosome rearrangements, although pericentric inversions are rare. Here we report a mother carrying a pericentric inversion of chromosome 14, and her daughter with recombinant chromosome characterized by a partial distal 14q trisomy. Principal clinical findings of the child include facial anomalies, microcephaly, developmental delay, hypotonia and cardiac malformation. Her final karyotype was 46,XX,rec(14)dup(14q)inv(14)(p12q31)mat[20], arr 14q31.3qter(85,427,839–106,356,482)x3. This report brings new data about clinical features of partial 14q trisomy and molecular analysis enables the visualization of genes involved in the segment duplicated.     

TTC-脱氢酶还原法测定螺旋藻细胞活性的条件优化

为确定氯化三苯基四氮唑(TTC)-脱氢酶还原法测定螺旋藻细胞活性的最优条件,首先通过单因素实验对影响藻细胞活性测定的TTC质量分数、缓冲液pH、提取剂（乙醇）质量分数、培养时间、培养温度进行分析,选定各因素的变化范围,再利用正交试验设计方法,在藻细胞活性测定的最适培养温度下,对TTC质量分数、缓冲液pH、提取剂质量分数、培养时间进行3水平优化试验。最后确定优化的TTC 脱氢酶还原法测定螺旋藻细胞活性的条件为TTC质量分数0.1%、缓冲液pH 8.0~8.5、乙醇质量分数60%、培养时间5 h、培养温度35 ℃。在此条件下所测藻细胞活性最高,为螺旋藻细胞活性的评价提供了一定参考。     

Tetratricopeptide Repeat Domain 9A Negatively Regulates Estrogen Receptor Alpha Activity

Tetratricopeptide repeat domain 9A (TTC9A) is a target gene of estrogen and progesterone. It is over-expressed in breast cancer. However, little is known about the physiological function of TTC9A. The objectives of this study were to establish a Ttc9a knockout mouse model and to study the consequence of Ttc9a gene inactivation. The Ttc9a targeting vector was generated by replacing the Ttc9a exon 1 with a neomycin cassette. The mice homozygous for Ttc9a exon 1 deletion appear to grow normally and are fertile. However, further characterization of the female mice revealed that Ttc9a deficiency is associated with greater body weight, bigger thymus and better mammary development in post-pubertal mice. Furthermore, Ttc9a deficient mammary gland was more responsive to estrogen treatment with greater mammary ductal lengthening, ductal branching and estrogen target gene induction. Since Ttc9a is induced by estrogen in estrogen target tissues, these results suggest that Ttc9a is a negative regulator of estrogen function through a negative feedback mechanism. This is supported by in vitro evidence that TTC9A over-expression attenuated ERα activity in MCF-7 cells. Although TTC9A does not bind to ERα or its chaperone protein Hsp90 directly, TTC9A strongly interacts with FKBP38 and FKBP51, both of which interact with ERα and Hsp90 and modulate ERα activity. It is plausible therefore that TTC9A negatively regulates ERα activity through interacting with co-chaperone proteins such as FKBP38 and FKBP51.     

Molecular characterization and functional analysis of a specific secreted protein from highly virulent defoliating Verticillium dahliae

Verticillium dahliae Kleb. is a phytopathogenic fungus that causes wilt diseases in hundreds of dicotyledonous plant species. Previous research has demonstrated that the secretome plays an important role in the pathogenicity of V. dahliae. In this study, the specific secreted protein gene (VdSSP1) in highly virulent defoliating V. dahliae strain VDG1 was cloned, and considered to be a secreted protein by signal peptide activity assay. VdSSP1 deletion mutants in VDG1 significantly compromised virulence, and the fungal growth decreased in media with pectin and starch as carbon sources. Pathogenicity and carbon utilization were restored upon complementation of the VdSSP1 deletion strains or low virulence non-defoliating strain VDG2, which lacks VdSSP1. It is indicated that the virulence role of VdSSP1 is associated with plant cell wall degradation. In conclusion, our data suggested that VdSSP1 is a secreted protein that is engaged in the pathogenicity of the highly virulent defoliating V. dahliae.     

Endophytic Pseudomonas pseudoalcaligenes shows better response against the Magnaporthe grisea than a rhizospheric Bacillus pumilus in Oryza sativa (Rice)

Two plant growth promoting rhizobacteria (PGPR), one identified as rhizospheric Bacillus pumilus and other as endophytic Pseudomonas pseudoalcaligenes, were isolated from the root surface as well as from within the roots of paddy variety GJ17. Adhesion and invasion of the isolated strains with the paddy root was confirmed by 2, 3, 5-triphenyl tetrazolium chloride (TTC) staining. The effects of these two PGPRs were tested alone and in combination on the production of defense related enzymes such as chitinase, polyperoxidase (PO) and polyphenol oxidase (PPO) in the presence of Magnaporthe grisea, the causative agent of rice blast. The results indicate that the endophytic bacteria showed a better response to the fungal infection than the rhizospheric one. The PGPRs were able to induce the defense enzymes even in the absence of the pathogen. This induction of defense enzymes in response to PGPRs persists for the entire life of the plant to defend against pathogens. So association of PGPRs with the paddy GJ-17 root acts as a vaccine to reduce disease severity by Magnaportha grisea.     

Rapid detection of Down's syndrome using quantitative real-time PCR (qPCR) targeting segmental duplications on chromosomes 21 and 11

Objective

Development of a qPCR test for the detection of trisomy 21 using segmental duplications.

Methods

Segmental duplications in the TTC3 gene on chromosome 21 and the KDM2A gene on chromosome 11 were selected as molecular markers for the diagnostic qPCR assay. A set of consensus primers selected from the conserved regions of these segmental duplications were used to amplify internal diverse sequences that were detected and quantified with different probes labeled with distinct fluorescence. The copy numbers of these two fragments were determined based on the ΔCq values of qPCR. The results of qPCR for prenatal and neonatal screening of Down's syndrome were compared with the conventional karyotype analysis by testing 82 normal individuals and 50 subjects with Down's syndrome.

Results

The ΔCq values of segmental duplications on chr21 and 11 ranged between 0.33 and 0.75 in normal individuals, and between 0.91 and 1.18 in subjects with Down's syndrome. The ΔCq values of these two segmental duplications clearly discriminated Down's syndrome from normal individuals (P < 0.001). Furthermore, the qPCR results were consistent with karyotype analysis.

Conclusion

Our qPCR can be used for rapid prenatal and neonatal screening of Down's syndrome.