Theoretical description of logical stochastic resonance and its enhancement: Fast Fourier transform filtering method.

Over the past decade, dynamic schemes have been proposed for the use of bistable systems in the design of logic devices. A bistable system in a noisy background can operate as a reliable logic gate in a moderate noise level, which is called a logical stochastic resonance (LSR). In this paper, we theoretically explore the emergence of LSR in general bistable systems and identify the dynamical mechanisms of LSR. The timescale relationship between the measured time and the mean first-pass time of two-state transitions is a key condition in determining whether the system is reliable. Furthermore, we demonstrate that the stability of the logic operation can be significantly improved by choosing the appropriate filtering method. Low-pass filtered noise-driven systems are more stable than Gaussian white noise. However, band-pass and high-pass filtered noise are harmful to the stability of the system due to the filtering of low-frequency components. Our theoretical and numerical simulation results offer perspectives for the development of logic devices.

Soyasaponin I alleviates hypertensive intracerebral hemorrhage by inhibiting the renin-angiotensin-aldosterone system.

BACKGROUND: Hypertensive intracerebral hemorrhage (HICH) is a life-threatening disease and lacks effective treatments. Previous studies have confirmed that metabolic profiles altered after ischemic stroke, but how brain metabolism changes after HICH was unclear. This study aimed to explore the metabolic profiles after HICH and the therapeutic effects of soyasaponin I on HICH. METHODS: HICH model was established first. Hematoxylin and eosin staining was used to estimate the pathological changes after HICH. Western blot and Evans blue extravasation assay were applied to determine the integrity of the blood-brain barrier (BBB). Enzyme-linked immunosorbent assay was used to detect the activation of the renin-angiotensin-aldosterone system (RAAS). Next, liquid chromatography-mass spectrometry-untargeted metabolomics was utilized to analyze the metabolic profiles of brain tissues after HICH. Finally, soyasaponin I was administered to HICH rats, and the severity of HICH and activation of the RAAS were further assessed. RESULTS: We successfully constructed HICH model. HICH significantly impaired BBB integrity and activated RAAS. HICH increased PE(14:0/24:1(15Z)), arachidonoyl serinol, PS(18:0/22:6(4Z, 7Z, 10Z, 13Z, 16Z, and 19Z)), PS(20:1(11Z)/20:5(5Z, 8Z, 11Z, 14Z, and 17Z)), glucose 1-phosphate, etc., in the brain, whereas decreased creatine, tripamide, D-N-(carboxyacetyl)alanine, N-acetylaspartate, N-acetylaspartylglutamic acid, and so on in the hemorrhagic hemisphere. Cerebral soyasaponin I was found to be downregulated after HICH and supplementation of soyasaponin I inactivated the RAAS and alleviated HICH. CONCLUSION: The metabolic profiles of the brains changed after HICH. Soyasaponin I alleviated HICH via inhibiting the RAAS and may serve as an effective drug for the treatment of HICH in the future.

A pilot study: Gut microbiota, metabolism and inflammation in hypertensive intracerebral haemorrhage.

AIMS: In recent years, the incidence rate of hypertensive intracerebral haemorrhage (HICH) has been increasing, accompanied by high mortality and morbidity, which has brought a heavy burden to the social economy. However, the pathogenesis of HICH is still unclear. This study intends to explore the mechanism of gut microbiota metabolism and inflammation in the process of HICH to provide a theoretical basis for the diagnosis and treatment of HICH. METHODS AND RESULTS: HE staining showed that the brain tissues of model group had obvious oedema injury, which indicated that the HICH model was successfully constructed. ELISA analysis showed that IL-1ß and TNF-α levels in blood and brain tissues were significantly increased, and IL-10 level was significantly decreased in blood. IHC analysis showed that microglia and macrophages were activated in the model group. 16S rRNA sequence showed that the diversity of gut microbiota in HICH patients decreased. Also, the microbiota belonging to Firmicutes, Proteobacteria and Verrucomicrobia changed significantly. LC-MS/MS analysis showed that the metabolic phenotype of HICH patients changed. Also, the 3,7-dimethyluric acid- and 7-methylxanthine-related metabolic pathways of caffeine metabolism pathways were downregulated in patients with HICH. Bacteroides was negatively correlated with the IL-1ß and TNF-α levels. Blautia was negatively correlated with the IL-1ß and TNF-α levels, and positively correlated with the IL-10 level. Akkermansia was negatively correlated with the 3,7-dimethyluric acid and 7-methylxanthine. CONCLUSION: Our study suggested that HICH was accompanied by the increased inflammation marker levels in peripheral blood and brain, decreased gut microbiota diversity, altered gut metabolic phenotype and downregulation of caffeine metabolism pathway. SIGNIFICANCE AND IMPACT OF THE STUDY: Our study reported that HICH accompanied by the increased inflammation, decreased gut microbiota diversity and altered gut metabolic phenotype. Due to the number of patients, this work was a pilot study.

4,5-Dimethoxycanthin-6-one is a novel LSD1 inhibitor that inhibits proliferation of glioblastoma cells and induces apoptosis and pyroptosis.

BACKGROUND: Glioblastoma is one of the most common fatal intracranial malignancies. Lysine-specific demethylase 1 (LSD1) reportedly has therapeutic effects on a variety of tumors. This study explored the therapeutic effect of LSD1 inhibition on glioblastoma cell lines and the possible underlying mechanisms. METHODS: The MTT assay was utilized to screen for the sensitivity of U87, U251 and T98G cells to 4, 5-dimethoxycarrageenin-6-one. qRT-PCR and western blot were used to measure the proliferation, apoptosis, and pyroptosis signaling pathway expression to observe the effect of LSD1 inhibition on U251 and T98G cells. Flow cytometry, immunofluorescence, immunohistochemistry, wound scratch, clone formation, and TUNEL assay were used to analyze the effects of 4, 5-dimethoxycanthin-6-one on glioblastoma cells. The effect of 4, 5-dimethoxycanthin-6-one was examined in vivo in BALB/c nude mice injected with U251 cells. HE staining was used to detect the histopathology of the tumor. RESULTS: LSD1 specifically catalyzes the demethylation of monomethylated and demethylated histone H3 lysine at position 4 (h3k4me1, h3k4me2, h3k4me3) and lysine at position 9 (h3k9me1). This regulated the transcriptional activity of proliferation, apoptosis, and pyroptosis signaling pathway genes. In vitro, the proliferation of glioblastoma cells was decreased in the 4, 5-dimethoxycanthin-6-one group. The expression of Caspase1 in glioblastoma cells treated with 4, 5-dimethoxycanthin-6-one increased, and the number of apoptotic cells increased. The tumor volume of mice injected with 4, 5-dimethoxycanthin-6-one decreased significantly. CONCLUSION: 4, 5-Dimethoxycanthin-6-one could act as a novel inhibitor of LSD1 to regulate glioblastoma, which could inhibit the proliferation of U251 and T98G cells and induce their apoptosis and pyroptosis. It is a potential drug for the treatment of glioblastoma.

A kinetic model of multiple phenotypic states for breast cancer cells.

Quantitative modeling of microscopic genes regulatory mechanisms in an individual cell is a crucial step towards understanding various macroscopic physiological phenomena of cell populations. Based on the regulatory mechanisms of genes zeb1 and cdh1 in the growth and development of breast cancer cells, we propose a kinetic model at the level of single cell. By constructing the effective landscape of underlying stationary probability for the genes expressions, it is found that (i) each breast cancer cell has three phenotypic states (i.e., the stem-like, basal, and luminal states) which correspond to three attractions of the probability landscape. (ii) The interconversions between phenotypic states can be induced by the noise intensity and the property of phenotypic switching is quantified by the mean first-passage time. (iii) Under certain conditions, the probabilities of each cancer cell appearing in the three states are consistent with the macroscopic phenotypic equilibrium proportions in the breast cancer SUM159 cell line. (iv) Our kinetic model involving the TGF-ß signal can also qualitatively explain several macroscopic physiological phenomena of breast cancer cells, such as the "TGF-ß paradox" in tumor therapy, the five clinical subtypes of breast cancer cells, and the effects of transient TGF-ß on breast cancer metastasis.

Salvia miltiorrhiza Bunge (Danshen) extract attenuates permanent cerebral ischemia through inhibiting platelet activation in rats.

ETHNOPHARMACOLOGICAL RELEVANCE: Danshen is a crude herbal drug isolated from dried roots of Salvia miltiorrhiza Bunge. This plant is widely used in oriental medicine for the treatment of cardiovascular and cerebrovascular diseases. The supercritical CO2 extract from Danshen (SCED) (57.85%, 5.67% and 4.55% for tanshinone IIA, tanshinone I and cryptotanshinone respectively) was studied in this article, whose potential molecular mechanism remains unclear, especially in anti-thrombosis. AIM OF THE STUDY: The present study was designed to observe the protective effect of SCED on ischemic stroke in rats and to explore the underlying anti-thrombosis mechanism. MATERIALS AND METHODS: Following induction of cerebral ischemia in rats by permanent middle cerebral artery occlusion (pMCAO). Neurological defect score, cerebral blood flow, infarct size, and brain edema were measured to evaluate the injury. Arteriovenous shunt thrombosis model and adenosine 5'-diphosphate (ADP) induced acute pulmonary embolism model were conducted to estimate the antithrombotic effect of SCED. In order to investigate the effects of SCED on platelet aggregation, rat platelet-rich-plasma (PRP) were incubated with SCED prior to the addition of the stimuli (ADP or 9, 11-dideoxy-11α, 9α-epoxymethanoprostaglandin F2α (U46619)). Aggregation was monitored in a light transmission aggregometer. Inhibitory effect of SCED on thromboxane A2 (TXA2) release was detected by ELISA kit. Phospholipase C (PLC)/ Protein kinase C (PKC) signaling pathway was analyzed by a Western blot technique. The effect of the SCED was also studied in vivo on bleeding time in mice. RESULTS: SCED improved the neurological defect score, increased cerebral blood flow, reduced infarct size and alleviated brain edema in rats exposed to pMCAO. After administration of SCED, thrombosis formation in arteriovenous shunt was inhibited and recovery time in pulmonary embolism was shortened. The inhibitory effect of SCED on platelet activation was further confirmed by TXB2 ELISA kit and Western blot analysis of PLC/PKC signaling pathway. CONCLUSIONS: SCED attenuates cerebral ischemic injury. The possible mechanism is that SCED inhibits thrombosis formation, platelet aggregation and activation of PLC/PKC pathway. On this basis, this new extract could be a promising agent to inhibit thrombosis formation and protect against cerebral ischemia injury.

Fluctuation and noise propagation in phenotypic transition cascades of clonal populations.

Quantitative modeling of fluctuations of each phenotype is a crucial step towards a fundamental understanding of noise propagation through various phenotypic transition cascades. The theoretical formulas for noise propagation in various phenotypic transition cascades are derived by using the linear noise approximation of master equation and the logarithmic gain. By virtue of the theoretical formulas, we study the noise propagation in bidirectional and unidirectional phenotypic transition cascades, respectively. It is found that noise propagation in these two phenotypic transition cascades evidently differs: In the bidirectional cascade, a systemic random environment is provided by a correlated global component. The total noise of each phenotype is mainly determined by the intrinsic noise and the transmitted noise from other phenotypes. The intrinsic noise enlarged by interconversion through an added part shows a novel noise propagation mechanism. However, in the unidirectional cascade, the random environment of each downstream phenotype is provided by upstream phenotypes. The total noise of each downstream phenotype is mainly determined by the transmitted noises from upstream phenotypes. The intrinsic noise and the conversion noise can propagate in both bidirectional and unidirectional phenotypic transition cascades.

[Effects of Down-regulating PPP2R5C Expression on Expression Profile of TAL1-related Regulating Genes in Jurkat Cells].

OBJECTIVE: Based on our previous study showing the inhibition of lenkemia T cell proliferation by down-regulating PPP2R5C expression, this study was aimed to analyze the influence of down-regulating PPP2R5 expression via RNA interference on genes relatied with TAL1 signaling pathway by using gene chip technique. METHODS: The PPP2R5C-siRNA799 was transduced into Jurkat cells by nucleofection, the total RNA was isolated from treated Jurkat cells after culture for 48 hours; the target sequences were prepared by revevse transcription after mRNA purification, and were hybridized with affymetrix gene expression profile chip 3' IVT. The original image data were collected using affymetrix gene chip scanner 3 000, and the gene expression profile was analyzed using gene spring GX 11.0 soflware. RESULTS: The expression of all 26 genes related with TAL1 signaling pathway was changed, out of which the expression of 15 genes were up-regulated and the expression of 11 genes was down-regulated in PPP2R5C-siRNA 799-transfected Jurkat cells. The genes with significantly up-regulated expression were GATA1, TCF4, XRCC6 and TCF3, while the genes with significantly down-regulated expression were SIN3A and RUNX1. CONCLUSION: The down-regulation of PPP2R5C gene expression in Jurkat cells via RNA interference to a certain degree can inhibit TAL1 signaling pathway genes, thereby suppresses the proliferation of Jurkat cells.

Fluctuations of cell population in a colonic crypt.

The number of stem cells in a colonic crypt is often very small, which leads to large intrinsic fluctuations in the cell population. Based on the model of cell population dynamics with linear feedback in a colonic crypt, we present a stochastic dynamics of the cell population [including stem cells (SCs), transit amplifying cells (TACs), and fully differentiated cells (FDCs)]. The Fano factor, covariance, and susceptibility formulas of the cell population around the steady state are derived by using the Langevin theory. In the range of physiologically reasonable parameter values, it is found that the stationary populations of TACs and FDCs exhibit an approximately threshold behavior as a function of the net growth rate of TACs, and the reproductions of TACs and FDCs can be classified into three regimens: controlled, crossover, and uncontrolled. With the increasing of the net growth rate of TACs, there is a maximum of the relative intrinsic fluctuations (i.e., the Fano factors) of TACs and FDCs in the crossover region. For a fixed differentiation rate and the net growth rate of SCs, the covariance of fluctuations between SCs and TACs has a maximum in the crossover region. However, the susceptibilities of both TACs and FDCs to the net growth rate of TACs have a minimum in the crossover region.

[One-year continuous observation of change in peripheral T cell subsets in workers exposed to low levels of benzene].

OBJECTIVE: To observe the T cell subsets and blood cells in the peripheral blood of workers exposed to low levels of benzene for one year, and to investigate the relationship between T cell function impairment and benzene-induced hematopoietic injury after benzene exposure. METHODS: Eighty-eight workers (58 males and 30 females, aged 18 ∼ 22 years) who just began to work in the workshop of a paint factory with exposure to benzene in Guangzhou, China were assigned to experimental group, and 88 workers (58 males and 30 females, aged 18 ∼ 25 years) who worked in the workshop without exposure to benzene were selected as controls. The blood samples of the workers were examined once every 4 months to measure the percentages of peripheral T cell subsets and peripheral blood cell counts in the one-year study. The benzene concentrations at operation points were also measured. RESULTS: The peripheral blood cell counts in the benzene-exposed workers had no significant changes in the first and second examinations; the white blood cell (WBC) counts in the experimental group in the third and fourth examinations were significantly lower than that in the control group [(6.4 ± 3.0)×10(9)/L and (6.3 ± 2.7)×10(9)/L vs (7.3 ± 3.0)×10(9)/L, P < 0.05], and the platelet (PLT) count in the experimental group in the fourth examination was also significantly lower than that in the control group[(179 ± 74)×10(9)/L vs (189 ± 70)×10(9)/L, P < 0.05]. Compared with those in the control group (CD4+: 54.29 ± 12.78%, CD8+: 37.25 ± 12.30%), the percentage of CD3+ T cells in the experimental group increased in the third examination; the percentage of CD4+ T cells in the experimental group decreased continuously in the second, third, and fourth examinations (50.77 ± 11.05%, 45.40 ± 9.41%, and 41.27 ± 10.62%), while the percentage of CD8+ T cells in the experimental group kept increasing (46.07 ± 10.18%, 50.36 ± 10.62%, and 56.40 ± 9.41%) (P < 0.05). CONCLUSION: The change in T cell subsets precedes that in the blood system in the workers exposed to low levels of benzene.

Observation of Microvascular Perfusion in the Hegu (LI4) Acupoint Area after Deqi Acupuncture at Quchi (LI11) Acupoint Using Speckle Laser Blood Flow Scanning Technology.

The aim of this study was to investigate the traditional meridian theory using speckle laser blood flow scanning technology to observe microcirculation of the Hegu acupoint area after acupuncture stimulation on distant points. An observational study was conducted to observe the microvascular perfusion of Hegu (LI4) and control points after acupuncturing Quchi (LI11). Thirty healthy volunteers (mean age 31.6 ± 8.7 years) received deqi acupuncture on Quchi (LI11, right side), and simultaneously changes in microvascular perfusion of Sanjian (LI3), Hegu (LI4), Yangxi (LI5), and two control points were observed before, during, and after needling using a MOOR speckle laser. The results showed that the changes in microvascular perfusion of the observed points are not regular. After correction, the experiment showed that the blood perfusion on 3 meridian acupoints was increased while the perfusion on 2 control points was decreased following acupuncture stimulation, the changes at Hegu (LI4) being the statistically most significant ones. Deqi acupuncture can help in regulating the body's blood flow, with a certain degree of meridian specificity.

[Change in the pattern of peripheral blood TCR V ß gene repertoire in occupational lead-exposed workers].

AIM: To investigate the distribution and clonality of TCR Vß subfamily in peripheral blood from workers exposed to lead, in order to understand the change in T cell immunity of occupational lead-exposed workers. METHODS: The CDR3 of TCR Vß 24 subfamily genes was amplified in peripheral blood mononuclear cells (PBMC) from 6 lead-exposed workers and 6 healthy individuals using RT-PCR, the positive PCR products were further subjected by genescan analysis to identify T cell clonality. RESULTS: All 24 TCR Vß subfamilies were detected in PBMC from 6 healthy individuals which all have polyclonal patterns. Only 1-7 Vß subfamilies could be identified in lead-exposed workers. In detected TCR Vß subfamilies, almost oligoclonal and biclonal patterns which mainly in Vß1 and Vß16. CONCLUSION: The restricted expression and clonal expansion of TCR Vß subfamily have been found in occupational lead-exposed workers, it may have some relationship with lead toxicity damage to the immune function.

[Establishment of stable subline of K562 cells expressing human leucocyte antigen a1101].

The aim of this study was to establish a stable subline of K562 cells expressing the HLA-A(*)1101 protein, which was expected to provide target cells for characterizing the HLA-I restrictive antigen specific cytotoxic T lymphocyte (CTL) effects against chronic myeloid leukemia (CML). The HLA-A(*)1101 protein encoding gene was amplified from peripheral blood mononuclear cell (PBMNC) of CML patient by RT-PCR; the 2A peptide linker (D-V-E-X-N-P-G-P) gene was linked to the 3'terminal of the HLA-A(*)1101 gene by recombinant PCR, then the recombinant was cloned into the pEGFP-N3 plasmid which contains an enhanced green fluorescent protein gene, and the eukaryotic recombinant expression vector containing HLA-A(*)1101-T2A-EGFP transcription box was constructed; the pEGFP-N3 vector and recombinant vector was separately electroporated into K562 cells. The expression of GFP was monitored by fluorescence microscopy, finally stably transfected sublines of K562 cells containing HLA-A(*)1101 gene, and of K562 containing pEGFP-N3 vector were obtained by G418 selection; the transcriptional or translational expression of HLA-A(*)1101 gene was detected with RT-PCR and flow cytometry respectively. The results indicated that the eukaryotic expression vector HLA-A(*)1101-T2A-EGFP plasmid was successfully constructed; after G418 selection for 2 months, two sublines of K562 cells (HLA-A(*)1101(+)K562, pEGFP-N3(+)K562) expressing GFP were constructed. The expression of HLA-A*A1101 gene could be determined in HLA-A(*)1101(+)K562 cell line by RT-PCR, while the pEGFP-N3(+)K562 cells could not express HLA-A*A1101 gene. HLA-A(*)1101 protein and GFP double positive HLA-A(*)1101(+)K562 cells were up to 88.5%, which was obviously higher than pEGFP-N3(+)K562 cells (0.698%) by flow cytometric analysis. It is concluded that a simple and effective method to select HLA-A(*)1101(+)K562 cells has been established and a subline of K562 cell expressing HLA-A(*)1101 protein on its cell membrane was successfully constructed, which provides the tool cells for further studying the specific cellular immunity against-CML.

[Analysis of TCR gene rearrangement for identification of T cell leukemia clone by using fine-tiling aCGH].

AIM: To establish a new method which analyzes T cell receptor (TCR) gene rearrangement for identification of T cells acute lymphoblastic leukemia (T-ALL) clone, it will provide the basis for the study of T-ALL including the chromosome translocation involving TCR loci. METHODS: Total DNA was isolated from peripheral blood mononuclear cells (PBMC) of one case with T-ALL. Using the fine-tiling array comparative genomic hybridization (fine-tiling aCGH) to analyze the genomic DNA differences of the case and control group, we could find the breakpoints and their position in the chromosomes. According to the preliminary results, we could design the specific primers for the positions of the breakpoints relative to sequence. Furthermore, the ligation-mediated PCR (LM-PCR) and sequence analysis were used to identify the TCR gene rearrangement. And TCR gene expression was detected by RT-PCR. RESULTS: The fine-tiling aCGH results of the T-ALL showed that the TCRα/δ locus of chromosome 14 appeared four breakpoints, corresponding to TCR Vδ1, Vδ2, Jδ1 and Jδ2. By LM-PCR, sequencing and sequence analysis, TCR gene of the case of T-ALL was involved in Vδ1Dδ2Dδ3Jδ1, Vδ2Dδ3Jδ2 rearrangement. RT-PCR results also confirmed the expression of these TCR gene rearrangements. CONCLUSION: The results demonstrated that fine-tiling aCGH and LM-PCR techniques could be used to identify the TCR gene rearrangement as one of the best perfect methods. And it was also a way to find some fusion genes involving in TCR gene.

[TCRζ gene expression in TCR gene-modified T cells specific for DLBCL-associated antigen].

AIM: To analyze the expression level of TCRζ chain gene in the DLBCL-associated antigen-specific T cells before and after being activated by coculture with Toledo cells (DLBCL cell line). METHODS: Real-time PCR with SYBR GreenI technique was used for detecting TCRζ chain expression in activated and unactivated DLBCL-associated antigen-specific T cells. ß2 microglobulin gene (ß2M) was used as an endogenous reference. Relative mRNA expression level of TCRζ gene was analyzed by the formula of both 2(-δCt); ×100% and 2(-δδCt);. RESULTS: Compared with (1.74±0.28)% of the relative mRNA expression level of TCRζ gene in TCR gene-untransduced T cells, the expression level of TCRζ gene was (1.78±0.22)% in unactivated TCR gene-transduced T cells and showed no obvious increase. While the expression of TCRζ gene arrived at (11.54±1.98)% in the activated TCR gene-modified T cells, which was significantly higher than that in unactivated TCR gene-modified and TCR gene-untransduced T cells (P<0.05), and was increased (6.59±0.80) and (6.48±0.36) times, respectively. CONCLUSION: The expression of TCRζ chain was up-regulated, when TCR gene-modified T cells were activated by the stimulation of specific antigens.

[Effect of superantigen SEA on the CD3epsilon chain expression on cord blood mononuclear cells stimulated by K562 cells].

AIM: To explore the effect of staphylococcal enterotoxin A(SEA) on CD3(epsilon) chain expression on mononuclear cells in cord blood, which were stimulated by K562 cells. METHODS: The anti-CD3 antibodies(mAb), K562 cells, SEA or both SEA and K562 cells were cocultured with mononuclear cells (MNCs) from four normal human cord blood for 48 hours respectively, Real-time PCR with SYBR Green I technique was used to detect expressed level of CD(epsilon) on MNCs in cord blood and set up a blank control group. Relative changes in expression level of CD3(epsilon) chain were indicated by the 2(-deltadeltaCt); method between each group and the control group. beta2-microglobulin gene(beta2M) was used as an endogenous reference. RESULTS: The expressed level of CD3(epsilon) chain on MNCs was slightly decreased in K562 cell group and were increased in the anti-CD3 antibodies mAb group, SEA group, both of SEA and K562 cell group respectively. The expressed level of CD3(epsilon) chain in both SEA and K562 group was significantly higher than that in the SEA group(P<0.01). CONCLUSION: Superantigen SEA can enhance the expressed level of CD3(epsilon) chain on MNCs stimulated by K562 cells.

[Change of expression pattern of CD3 genes in peripheral blood T-cells from CML patients].

Our previous finding showed that down-regulation of CD3ζ gene was detected in patients with chronic myeloid leukemia (CML). In order to further elucidate the feature of T cell immune status in the signal transduction in CML patients, the expression patterns of all 4 CD3 genes were characterized in peripheral blood of patients, the expression levels of CD3γ, δ, ε and ζ chain genes were detected by real time qPCR with SYBR Green I staining in peripheral blood mononuclear cells (PBMNCs) from 17 cases of de novo CML patients in chronic phase and 17 cases of healthy individuals, the ß2-microglobulin gene was used as an internal reference, and the mRNA expression level of each CD3 gene was evaluated by the 2(-ΔCt) x 100% method. The results showed that the median expression levels of CD3γ, δ and ε genes (2.344%, 0.515% and 3.516%) in CML patients were not significantly different from healthy individuals (p = 0.072, p = 0.190, p = 0.615, respectively), while the expression level of CD3ζ gene in PBMNCs from CML patients (0.395%) was lower than that from healthy individuals (1.538%) (p < 0.001). The expression patterns of 4 CD3 genes in proper order were CD3ε > CD3γ > CD3δ > CD3ζ in CML group, in contrast, the expression patterns were presented as CD3γ > CD3ε > CD3ζ > CD3δ in healthy group. It is concluded that the present study characterized the expression pattern of CD3γ, δ, ε and ζ chain genes in CML patients, lower expression of CD3ζ is the feature of TCR signal transduction immunodeficiency and the expression patterns of 4 CD3 genes are changed in CML patients.

[Structural feature and biological function of PPP2R5C gene].

PPP2R5C is one of the members of regulatory subunits of protein phosphatase 2A (PP2A), which plays a critical role in cell proliferation, differentiation and transformation, based on its induction of dephosphorylation of P53 at various residues. Recently, it was characterized that the alteration of expression pattern of PPP2R5C is associated with cell malignant transformation, thus PPP2R5C was thought as a marker for progressive disease in B-CLL. In this article the gene structure and biological function of PPP2R5C as well as relation of PPP2R5C with genesis and development of cancer were discussed.

[Analysis of CDR3 sequence of TCR Valpha6, Valpha10 and Vbeta23 oligoclonal T cells in one APL case].

AIM: To investigate the clonality and the pattern of CDR3 sequence of TCR Valpha and Vbeta repertoire in patients with acute promyelocytic leukemia (APL). METHODS: The CDR3 of TCR Valpha 29 and Vbeta 24 subfamily genes were amplified in mononuclear cells from peripheral blood of one case with APL using RT-PCR. The positive PCR products were further labeled with fluorescent and analyzed by genescan technique for the CDR3 size, to evaluate clonality of the detectable TCR Valpha and Vbeta T cells. The products of the oligoclonal T cells were analyzed by sequencing to define the sequence of CDR3. RESULTS: Oligoclonal T cells expressed TCR Valpha6, Valpha10 or Vbeta23 genes were identified from the APL patient. The molecular characteristics of the CDR3 of Valpha and Vbeta genes rearrangement were Valpha6NJalpha17, Valpha10NJalpha35 and Vbeta23NDbeta1NJbeta2.7. The amino acid sequence of CDR3 region in TCR Valpha 6, Valpha10 and Vbeta23 subfamilies were CAMRENSAGNK, YLCAGDELLWECA or CASSSKWAGGTYEQY, respectively. These sequences were accepted by GenBank (Accession Number EU544946, EU544947 and EU647219). CONCLUSION: Three idiotype Valpha and Vbeta sequences are identified in a case with APL, which is thought that may mediate the host specific immunity response for leukemia cells in APL. It might provide data for farther research on the specific immunotherapy of APL.

[Construction and expression of idiotype TCR Valpha1-pIRES-TCR Vbeta8 eukaryotic vectors in vitro].

AIM: To construct the eukaryotic vectors with idiotype TCR Valpha1-pIRES-TCR Vbeta8 of Jurkat cell line and investigate their expression in vitro after transferred into eukaryotic cells. METHODS: TCR Valpha and Vbeta subfamilies of Jurkat cells were analyzed by using RT-PCR and genescan technique. Then the monoclonal TCR Valpha1 and Vbeta8 genes of Jurkat cells were cloned into multiple clone site (MCS) A and MCS B of the eukaryotic vector pIRES respectively. Its sequences were identified by restriction enzyme cutting and sequence analysis. The expression of TCR mRNA and idiotypic protein in transferred A549 and Molt4 cells was tested by RT-PCR, indirect immunophenotyping fluorescein dyeing and flow cytometry, respectively. RESULTS: Two recombinavot eukaryotic vectors with idiotype TCR Valpha1-pIRES-TCR Vbeta8 were developed successfully and the expression of both idiotypic TCR mRNA and protein was detected in transferred A549 and Molt4 cells. CONCLUSION: The successful construction of the two eukaryotic vectors with idiotype TCR Valpha1-pIRES-TCR Vbeta8 will provide a basic method for further study of the specific TCR gene modified T cells.