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T-cell-engaging bispecific antibodies (BiTEs), which can simultaneously bind to antigens on tumor cells and T cells, show good potential in cancer immunotherapy. A practical and feasible approach for emulating BiTEs involves immobilizing two types of monoclonal antibodies (mAbs) onto a single nanoparticle; however, this approach involves complex immobilization processes and chemical reactions. To overcome these challenges, we achieved gentle antibody immobilization through receptor-ligand interactions by constructing a mAb delivery system known as Fcγ receptor 1 (FcγR1)-expressing cell membrane-coated nanoparticles (abbreviated as FcγR1-CMNPs). To validate the functionality and feasibility of this approach, we immobilized αCD3 and αCD20 onto the CMNP surface and generated a bispecific nanoantibody (termed CMNP@CD3 × CD20) for treating B-cell lymphoma. We demonstrated that CMNP@CD3 × CD20 significantly promoted the interaction between T cells and tumor cells and resulted in the potent killing of tumor cells in vitro and in vivo. This innovative antibody delivery system offers a straightforward and gentler technique for antibody delivery, potentially facilitating the development of trispecific or multispecific antibody-based immunotherapy.

The functions of natural killer (NK) and T cells in innate and adaptive immunity, as well as their functions in tumor eradication, are complementary and intertwined. Here we show that utilization of multi-specific antibodies or nano-antibodies capable of simultaneously targeting both NK and T cells could be a valuable approach in cancer immunotherapy. Here, we introduce a tri-specific Nano-Antibody (Tri-NAb), generated by immobilizing three types of monoclonal antibodies (mAbs), using an optimized albumin/polyester composite nanoparticle conjugated with anti-Fc antibody. This Tri-NAb, targeting PDL1, 4-1BB, and NKG2A (or TIGIT) simultaneously, effectively binds to NK and CD8 + T cells, triggering their activation and proliferation, while facilitating their interaction with tumor cells, thereby inducing efficient tumor killing. Importantly, the antitumor efficacy of Tri-NAb is validated in multiple models, including patient-derived tumor organoids and humanized mice, highlighting the translational potential of NK and T cell co-targeting., (© 2024. The Author(s).)

Background: Methamphetamine (MA) use increases the risk of age-related diseases. However, it remains uncertain whether MA use exhibits accelerated biological aging, as indicated by telomere length (TL), a proposed marker of aging. Here we conducted studies in both humans and rats to investigate the association between MA use and TL., Methods: We recruited 125 male MA users and 66 healthy controls, aged 30-40 years. MA users were diagnosed using DSM-5 criteria and categorized into two groups: non-severe (n = 78) and severe (n = 47) MA use disorder (MUD). MA-treated conditioned place preference (CPP) rats were utilized to validate our clinical investigations. TL was assessed using real-time polymerase chain reaction., Results: At clinical levels, MA users exhibited significantly shorter leukocyte TL compared to healthy controls. Among MA users, individuals with severe MUD had significantly shorter leukocyte TL than those with non-severe MUD. Importantly, both univariate and multivariate linear regression analyses demonstrated a negative association between the severity of MA use and leukocyte TL. In a rat model of MA-induced CPP, leukocyte TL was also significantly shortened after MA administration, especially in rats with higher CPP expression or reinstatement scores., Conclusion: MA use shortened TL, and the severity of MA use was negatively correlated with TL. These findings provide new insights into the pathophysiology of accelerated aging caused by MA use and may have implications for identifying biomarkers and developing novel treatment strategies for MUD., Competing Interests: Declaration of Competing Interest The authors declare no conflicts of interest., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Zanthoxylum armatum DC. is an important medicinal plant, and its pericarps are commonly used as a natural spice in Asian countries. In this study, fifteen alkylamides were isolated and elucidated from the pericarps of Z. armatum, including five undescribed alkylamides (1-5) and ten known compounds (6-15). The molecular structures of all compounds were elucidated by 1D and 2D NMR spectroscopic analysis and mass spectrometry, among which the absolute configuration of compound 15 was determined by the Mo 2 (OAc) 4 -induced circular dichroism method. Moreover, all compounds were screened for their neuroprotective activity against H 2 O 2 -induced oxidative stress in human neuroblastoma SH-SY5Y cells for the evaluation of their neuroprotective activity. Especially, compounds 2-4 expressed potential neuroprotective activity, and further research showed that the cell viability was significantly enhanced in a concentration dependent manner when the cells were treated for 6 h. Moreover, compounds 2-4 could decrease reactive oxygen species accumulation. This paper enriched structure types of alkylamides in Zanthoxylum armatum., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Zanthoxylum belongs to the Rutaceae family, and there are 81 Zanthoxylum species and 36 varieties in China. Most of the Zanthoxylum plants are used as culinary spice. In recent years, scholars in China and abroad have carried out in-depth research on Zanthoxylum plants, and found that the peculiar numbing sensation of Zanthoxylum plants originates from amides. It is also determined that amides are an important material basis for exerting pharmacological effects, especially in anti-inflammatory analgesia, anesthesia and other aspects. In this paper, 123 amides in 26 Zanthoxylum plants and their pharmacological activity that have been reported were summarized, which provided scientific reference for the clinical application of Zanthoxylum plants and the research and development of new drugs, and also facilitated the sustainable development and utilization of Zanthoxylum plant resources.

Immune cells are indispensable defenders of the human body, clearing exogenous pathogens and toxicities or endogenous malignant and aging cells. Immune cell dysfunction can cause an inability to recognize, react, and remove these hazards, resulting in cancers, inflammatory diseases, autoimmune diseases, and infections. Immune cells regulation has shown great promise in treating disease, and immune agonists are usually used to treat cancers and infections caused by immune suppression. In contrast, immunosuppressants are used to treat inflammatory and autoimmune diseases. However, the key to maintaining health is to restore balance to the immune system, as excessive activation or inhibition of immune cells is a common complication of immunotherapy. Nanoparticles are efficient drug delivery systems widely used to deliver small molecule inhibitors, nucleic acid, and proteins. Using nanoparticles for the targeted delivery of drugs to immune cells provides opportunities to regulate immune cell function. In this review, we summarize the current progress of nanoparticle-based strategies for regulating immune function and discuss the prospects of future nanoparticle design to improve immunotherapy., Competing Interests: Conflicts of interests: Authors state no conflict of interest., (© 2023 the author(s), published by De Gruyter, Berlin/Boston.)

Cleft palate is a common maxillofacial congenital malformation, and its mechanism still has not been fully illustrated. Recently, lipid metabolic defects have been observed in cleft palate. Patatin-like phospholipase domain-containing 2 (Pnpla2) is an important lipolytic gene. However, its effect on the formation of cleft palate remains unknown. In this research, we explored the expression of Pnpla2 in the palatal shelves of control mice. We also studied mice with cleft palates induced by retinoic acid and its effect on the embryonic palatal mesenchyme (EPM) cells phenotype. We found that Pnpla2 was expressed in the palatal shelves of both the cleft palate and control mice. Pnpla2 expression was lower in cleft palate mice than in the control mice. Experiments with EPM cells showed that knockdown of Pnpla2 inhibited cell proliferation and migration. In conclusion, Pnpla2 is linked to palatal development. We have indicated that low expression of Pnpla2 affects palatogenesis by inhibiting the proliferation and migration of EPM cells., (Copyright © 2023 The British Association of Oral and Maxillofacial Surgeons. Published by Elsevier Ltd. All rights reserved.)

Objective: To investigate the effects of different prescription compositions of traditional Chinese medicine and its different extraction methods of compound formula extracts on hypoxia tolerance in mice, in order to preferably select their prescription compositions and preparation extraction methods. Methods: Male BALB/c mice were randomly divided into 6 groups: blank control group, compound danshen group, compound Rhodiola Rosea alcohol-water extract group (Rhodiola rosea, Astragali Radix, Polygonati Rhizoma, Lycii Fructus), compound Rhodiola Rosea water extract group, compound Astragalus alcohol-water extract group (Astragali Radix, Polygonati Rhizoma, Lycii Fructus) and compound Astragalus water extract group, 30 mice in each group. Each group was administered continuously by gavage for 10 d. The blank group was gavaged with sterilized injection water. The mice in the other groups were treated with 0.15 g/kg of compound danshen, 3 g/kg of compound Rhodiola Rosea alcohol-water extract or water extract, and 1.7 g/kg of compound Astragalus alcohol-water extract or water extract, respectively. Each group was subjected to normobaric hypoxia tolerance test, sodium nitrite toxicity survival test and acute cerebral ischemia-hypoxia test 1 h after the last gavage, and the mice brain tissues were used to determine the activity of antioxidant enzymes and metabolites related to oxidative stress. Results: Compared with the blank control group, in normobaric hypoxia tolerance test, the survival time of mice in the compound danshen group and the compound Astragalus alcohol-water extract group and water extraction group was prolonged significantly ( P ＜0.01), and the number of open-mouth gasping after cerebral ischemia and hypoxia was increased significantly ( P ＜0.05). There was no statistical difference in survival time after sodium nitrite injection in each group. Compared with the blank control group, the activities of T-AOC, SOD, GSH and CAT were increased significantly ( P ＜0.05, P ＜0.01) and the content of MDA was decreased significantly ( P ＜0.01) in the compound Astragalus water extract group. Compared with the compound danshen group, the activities of SOD, CAT and GSH were increased significantly ( P ＜0.01, P ＜0.05) and the content of MDA was decreased significantly ( P ＜0.05). Conclusion: Compound Astragalus water extraction has the best effect of hypoxia tolerance, compound Rhodiola Rosea can eliminate Rhodiola rosea and consists of Astragali Radix, Polygonati Rhizoma, Lycii Fructus and its extraction method is water extraction.

Abnormally high concentrations of all-trans retinoic acid (atRA) induce cleft palate, which is accompanied by abnormal migration and proliferation of mouse embryonic palatal mesenchyme (MEPM) cells. Hormone-sensitive lipase (HSL) is involved in many embryonic development processes. The current study was designed to elucidate the mechanism of HSL in cleft palate induced by atRA. To establish a cleft palate model in Kunming mice, pregnant mice were administered atRA (70 mg/kg) by gavage at embryonic Day 10.5 (E10.5). Embryonic palates were obtained through the dissection of pregnant mice at E15.5. Hematoxylin and eosin (H&E) staining was used to evaluate growth changes in the palatal shelves. The levels of HSL in MEPM cells were detected by immunohistochemistry, quantitative real-time reverse transcription-polymerase chain reaction (qRT-PCR) and western blotting. RNAi was applied to construct vectors expressing HSL small interference RNAs (siRNAs). The vectors were transfected into MEPM cells. Cell proliferation and migration were evaluated by the cell counting kit-8 (CCK-8) assay and wound healing assay, respectively. The palatal shelves in the atRA group had separated at E15.5 without fusing. In MEPM cells, the expression of HSL was reversed after atRA treatment, which caused cleft palate in vivo . In the atRA group, the proliferation of HSL siRNA-transfected cells was remarkably promoted, and the migration rate significantly increased in the HSL siRNA-transfected MEPM cells. These results suggested that HSL may be involved in cleft palate induced by atRA and that atRA enhances HSL levels to inhibit embryonic palate growth.

Objective: The present study was to explore the feasibility of developing positron molecular probes for the metastasis-associated lung adenocarcinoma transcript 1 (MALAT-1), to evaluate the distribution and pharmacokinetics, and to explore whether the probe can be used for the imaging of malignant tumours with high MALAT-1 expression in vivo., Methods: [68Ga]Ga labelling of MALAT-1 antisense oligonucleotides ([68Ga]Ga-MALAT-1-ASO) was synthesized by the conjugation of MALAT-1-NOTA-ASO and [68Ga] Ga3+. The radiochemical purity was shown by radio-HPLC. Pharmacokinetic studies and cellular uptake studies were performed. The biodistribution and metabolism of [68Ga] Ga-MALAT-1-ASO in normal ICR and MHCC-LM3 xenograft-bearing nude mice were studied in vitro and in vivo., Results: [68Ga]Ga-MALAT-1-ASO was obtained in 98% radiochemical yield from a 10-min synthesis with 100 ± 50 MBq/nmol specific activity and >99% radiochemical purity. The Log D was -2.53 ± 0.19. The tracer displayed excellent stability in vitro. 68Ga-MALAT-1 ASO showed satisfactory binding ability to MHCC-LM3 cells; the biodistribution of [68Ga]Ga-MALAT-1-ASO in MHCC-LM3 tumour-bearing mice demonstrated specific uptake of the radiotracer (3.04 ± 0.11%ID/g). Micro-PET images of the MHCC-LM3 cell xenograft mouse model provided further evidence to support the hypothesis that [68Ga]Ga-MALAT-1-ASO can target tumours in vivo., Conclusions: We conclude that [68Ga]Ga labelling of MALAT-1 ASO is a convenient approach. The high accumulation of [68Ga]Ga-MALAT-1-ASO for tumours expressing MALAT-1 suggests that this radio compound may be used as a potential positron molecular probe. Molecular structure optimization studies need to be more in-depth to further reduce its background uptake and enhance tumour targeting., (Copyright © 2021 Wolters Kluwer Health, Inc. All rights reserved.)

In order to investigate the effect of salidroside on inhibiting liver fibrosis and its relationship with CXC chemokine ligand 16(CXCL16) in vivo and in vitro, totally 45 C57 BL/6 J male mice were randomly divided into normal group, model group and salidroside group, with 15 mice in each group. The mice in model group and salidroside group were injected intraperitoneally with 15% carbontetrachloride(CCl_4) olive oil solution to establish liver fibrosis model, and the mice in normal group were injected intraperitoneally with the same dose of olive oil. Salidroside group was given with 100 mg·kg~(-1 )salidroside by gavage, while the normal group and model group received the same amount of double distilled water by gavage. All mice were sacrificed after 5 weeks of intragastric administration. The pathological changes of mouse liver were observed by hematoxylin-eosin(HE) staining, and the degree of liver fibrosis was observed by sirius red staining. The protein expressions of collagen Ⅰ(ColⅠ), α-smooth muscle actin(α-SMA), fibronectin(FN), CXCL16, phosphorylated Akt(p-Akt), Akt in liver tissues were detected by Western blot. Hepatic stellate cell line JS 1 was cultured in vitro and divided into control group, model group(100 μg·L~(-1) CXCL16) and salidroside group(100 μg·L~(-1) CXCL16+1×10~(-5) mol·L~(-1) salidroside). Cell migration was detected by cell scratch, the mRNA expressions of ColⅠ and α-SMA were detected by RT-PCR, and the protein expressions of p-Akt and Akt were detected by Western blot. As compared with the normal group, the protein expressions of ColⅠ, α-SMA, FN, CXCL16, and p-Akt in the model group were significantly increased, and salidroside could reduce the expression of these indicators(P<0.05 or P<0.01). In vitro, CXCL16 could promote the migration of JS 1, increase the mRNA expressions of ColⅠ and α-SMA in JS 1, and enhance Akt phosphorylation in JS 1(P<0.05 or P<0.01). As compared with the model group, salidroside could inhibit the migration of JS 1 induced by CXCL16(P<0.05), and reduce the high expression of ColⅠ and α-SMA mRNA and the phosphorylation of Akt in JS 1 induced by CXCL16(P<0.05). In conclusion, salidroside might attenuate CCl_4-induced liver fibrosis in mice by inhibiting the migration, activation and Akt phosphorylation of hepatic stellate cells induced by CXCL16.

Considerable breakthroughs in the treatment of malignant tumors using antibody drugs, especially immunomodulating monoclonal antibodies (mAbs), have been made in the past decade. Despite technological advancements in antibody design and manufacture, multiple challenges face antibody-mediated cancer therapy, such as instability in vivo, poor tumor penetration, limited response rate, and undesirable off-target cytotoxicity. In recent years, an increasing number of biomaterials-based delivery systems have been reported to enhance the antitumor efficacy of antibody drugs. This review summarizes the advances and breakthroughs in integrating biomaterials with therapeutic antibodies for enhanced cancer therapy. A brief introduction to the principal mechanism of antibody-based cancer therapy is first established, and then various antibody immobilization strategies are provided. Finally, the current state-of-the-art in biomaterials-based antibody delivery systems and their applications in cancer treatment are summarized, highlighting how the delivery systems augment the therapeutic efficacy of antibody drugs. The outlook and perspective on biomaterials-based delivery of antitumor antibodies are also discussed., (© 2021 Wiley-VCH GmbH.)

Modulating effector immune cells via monoclonal antibodies (mAbs) and facilitating the co-engagement of T cells and tumor cells via chimeric antigen receptor- T cells or bispecific T cell-engaging antibodies are two typical cancer immunotherapy approaches. We speculated that immobilizing two types of mAbs against effector cells and tumor cells on a single nanoparticle could integrate the functions of these two approaches, as the engineered formulation (immunomodulating nano-adaptor, imNA) could potentially associate with both cells and bridge them together like an 'adaptor' while maintaining the immunomodulatory properties of the parental mAbs. However, existing mAbs-immobilization strategies mainly rely on a chemical reaction, a process that is rough and difficult to control. Here, we build up a versatile antibody immobilization platform by conjugating anti-IgG (Fc specific) antibody (αFc) onto the nanoparticle surface (αFc-NP), and confirm that αFc-NP could conveniently and efficiently immobilize two types of mAbs through Fc-specific noncovalent interactions to form imNAs. Finally, we validate the superiority of imNAs over the mixture of parental mAbs in T cell-, natural killer cell- and macrophage-mediated antitumor immune responses in multiple murine tumor models.

Exosomes are small vesicles with a bilayer membrane structure secreted by a variety of cells. They are widely distributed in a variety of body fluids and contain proteins, nucleic acids and other components. They can mediate information transmission between cells and participate in a variety of physiological and pathological activities of cells. Hepatocytes, hepatic sinus endothelial cells and other cells were able to communicate with hepatic stellate cells via exosomes, and regulate the activation, migration, apoptosis and other biological activities of hepatic stellate cells. In this review, the recent advances in the regulation of exosomes on the biological activity of hepatic stellate cells were reviewed.

Background: The prevalence of HIV among men who have sex with men (MSM) has become a significant public health challenge. The aim was to comprehensively estimate the national prevalence of HIV among MSM and its time trends through a large-scale systematic analysis., Methods: Systematic search of Cochrane Library, PubMed, EMBASE, CNKI, VIP, and Wanfang Data databases without language restriction for studies on the prevalence of HIV among MSM published before Dec.31, 2018. Studies were eligible for inclusion if they were published in the peer-reviewed literature and used validated assessment methods to assess the prevalence of HIV among MSM. Estimates were pooled using random-effects analysis., Results: Data were extracted from 355 cross-sectional studies (571,328 individuals) covered 59 cities from 30 provinces and municipalities of China. The overall national prevalence of HIV among MSM from 2001 to 2018 was estimated to be 5.7% (95% CI: 5.4-6.1%), with high between-study heterogeneity (I 2  = 98.0%, P <  0.001). Our study showed an increased tendency in the HIV prevalence as time progressed by meta-regression analysis (I 2  = 95.9%, P <  0.0001). HIV prevalence was the highest in those aged 50 years and older with HIV prevalence of 19.3% (95%CI: 13.1-27.4%, N = 13). HIV was more prevalent in the illiterate population (16.8%), than in those who had received an education. Although the internet was a major venue for Chinese MSM seeking male sex partners (35.6, 95%CI: 32.3-39.9%, N = 101), seeking MSM in bathhouses/saunas had the highest associated prevalence of HIV (13.4, 95%CI: 10.3-17.1%, N = 22). The HIV prevalence among MSM varied by location: compared with other regions in China, HIV was highly prevalent among MSM in the southwest (10.7, 95%CI: 9.3-12.2%, N = 91). Compared to participants who sometimes or always used condoms, participants who had never used a condom in the past 6 months had a higher risk of HIV infection, with odds ratios of 0.1 (95%CI: 0.08-0.14)., Conclusions: Our analysis provided reliable estimates of China's HIV burden among MSM, which appears to present an increasing national public health challenge. Effective government responses are needed to address this challenge and include the implementation of HIV prevention.

Aim: To clone prokaryotic expression vector of Cdc25C, purify the fusion protein of GST-Cdc25C, and identify its function preliminarily., Methods: Human Cdc25C coding region was amplified from human mammary cDNA library by PCR, and cloned into the prokaryotic expression vector pGEX-KG. The fusion protein GST-Cdc25C was expressed in E.coli Rossate and purified by GST-Sepharose 4B beads. The function of purified GST-Cdc25C was identified by GST pull-down assay., Results: The GST-Cdc25C recombinant plasmid was successfully obtained by double digestion identification. The inserted fragment was confirmed correctly by sequencing. SDS-PAGE and Western blot analysis showed that the fusion protein was expressed. The fusion protein of about M(r); 80 000 was successfully induced, and identified by SDS-PAGE and Western blot analysis. GST pull-down assay showed that GST-Cdc25C could interact with Chk2 which verified its known function., Conclusion: Cdc25C was successfully cloned and purified.

Aim: To prepare anti-PES1 monoclonal antibody (mAb) and detect the PES1 expression in several kinds of human cancer cell lines and its tissue distribution in the adult rat., Methods: pGEX-PES1(1-322aa) fusion protein was purified and inject-ed into mice for immunization. Anti-PES1 mAb was produced by cell fusion and screening after immunization. Anti-PES1 mAb was identified by Western blot. Several human cancer cell lines and different tissue samples of adult rat were detected the PES1 expressions with the mAb prepared., Results: Aanti-PES1 mAb was determined to be specific to PES1 with Western blot analysis. PES1 were expressed in all kinds of breast, ovary, liver and lung cancer cell lines detected in different levels with prepared mAb. Pescadillo was obviously expressed in the breast and ovary but not other tissues of adult rat using prepared mAb., Conclusion: Anti-PES1 mAb was successfully prepared. PES1 may play an important role in the tumorigenicity and may also play a role in the pathway of estrogen since breast and ovary, the most important estrogen target organ of adult rat, obviously express pesca-dillo.

Objective: To investigate whether progesterone receptor B (PRB) can be sumoylated by SUMO-2/3 and the effect of sumoylation on PRB transcriptional activity., Methods: SUMO-2/3 cDNA was amplified from MCF-7 cDNA and cloned into the eukaryotic expression vector pcDNA3-FLAG. The plasmid pXJ40-myc-PRB was cotransfected with pcDNA3FLAG-SUMO2, pcDNA3FLAG-SUMO3 or the mock control into 293T cells, and PRB sumoylation was detected by immunoprecipitation and Western blotting. The effect of PRB sumoylation on its transcriptional activity was determined using reporter luciferase assay., Results: pcDNA3FLAG-SUMO2 and pcDNA3FLAG-SUMO3 vectors were successfully constructed. SUMO-2/3 could bind covalently to PRB and increase its transcriptional dependent on the presence of progesterone., Conclusion: PRB can be sumoylated by SUMO-2/3 and its function is regulated by this modification.

Aim: To construct the prokaryotic expression vector of human histone-1, obtain the purified GST-H1 protein, and detect its activity., Methods: Human histone-1 coding region was amplified from human mammary cDNA library, and was inserted into prokaryotic expression vector pGEX-KG. The recombinant plasmid pGEX-KG-H1 was transformed into E.coli Rossate. The expressed product was purified by GST-Sepharose 4B beads and identified by SDS-PAGE and Western blot analysis., Results: The DNA fragment of about 650 bp was successfully amplified by PCR, cloned into pGEX-KG, and identified by sequencing. The recombinant protein of about M(r); 52 000 was successfully induced, purified and tested well by Kinase assay., Conclusion: The recombinant protein of GST-H1 is obtained successfully, which lay the foundation for further research on cell cycle control.

Aim: To construct a vector of ErbB2 small interfering RNA(siRNA), and to investigate its effect on ErbB2 expression and the growth of ZR75-1 breast cancer cells., Methods: Two ErbB2-siRNAs were designed and inserted into the pSliencer 2.1-U6 neo vector. After confirmed by restriction and DNA sequencing, siRNA vectors were co-transfected with the FLAG-ErbB2 expression vector into human embryonic kidney 293T cells, or transfected into SKBR3 and ZR75-1 breast cancer cells. The effects of siRNAs on the expression of ErbB2 were identified by Western blot and the proliferation of ZR75-1 cells was repressed., Results: Two siRNAs could effectively inhibit the expression of exogenous and endogenous ErbB2 proteins, and could repress the growth of ZR75-1 cells., Conclusion: ErbB2 siRNAs effectively inhibit the expression of ErbB2, thus repressing the growth of ZR75-1 cells.

Aim: To construct the eukaryotic expression vector of small interfering RNA (siRNA) targeting human FHL2 and transfect it into human embryo kidney 293T cells to investigate the silencing effect of FHL2 siRNA on the expression of FHL2 gene., Methods: Two FHL2 siRNAs were designed and inserted into pSliencer 2.1-U6 neo expression vector. Then human embryo kidney 293T cells were cotransfected with the recombinant plasmids and FLAG-tagged FHL2 expression vector. The silencing effect of FHL2 siRNAs on the expression of FHL2 gene was identified by Western blot., Results: The expression vectors of FHL2 siRNAs were constructed and confirmed by DNA sequencing. Western blot showed that FHL2 siRNAs effectively inhibited expression of FHL2., Conclusion: The eukaryotic expression vectors of FHL2 siRNAs are constructed successfully. The siRNAs effectively inhibit the expression of FHL2.

Estrogen receptor alpha (ERalpha) plays an important role in breast cancer development and progression and thus becomes a useful molecular target for breast cancer therapy. ERalpha is differentially expressed in breast cancer patients. Moreover, ERalpha expression levels may change at different stages of breast cancer even for the same patient. ERalpha expression is closely associated with the effect of endocrine therapy and prognosis. The mechanisms underlying ERalpha expression are complicated, because ERalpha expression is regulated at different levels, including chromatin, transcriptional, post- transcriptional, translational, and post-translational levels. Many proteins modulate the transcription of ERalpha gene at the chromatin and transcriptional levels through direct or indirect interaction with the ERa promoter. Some microRNAs decrease ERalpha levels possibly by induction of the degradation of ERalpha mRNA and/or repression of the mRNA translation. At the post-translational level, many proteins regulate ERalpha protein levels through ubiquitin-proteosome pathway. This review focuses on molecular mechanisms of regulation of ERalpha expression at different levels.

Aim: To construct human Egr-1 promoter luciferase reporter system and study its activity induced by ionizing radiation., Methods: Egr-1 promoter was obtained by human genomic PCR and cloned into pGL3-basic vector. After transfection of recombinant plasmid into human tumor cells, the Egr-1 promoter activity induced by ionizing radiation was detected by luciferase reporter assay., Results: The luciferase reporter system of Egr-1 promoter was successfully constructed. The activity of Egr-1 promoter was substantially increased after different doses of IR and reached to the peak at the time point of 48 h after IR., Conclusion: The Egr-1 promoter was constructed in this study showed IR inducible activity in tumor cells, laying foundation for the research of radiation. mediated gene therapy.

Objective: To identify the expression of Drosophila Eyes Absent Homologue 2 (EYA2) in non-small cell lung cancer (NSCLC) and to investigate its correlation with clinical parameters., Methods: 59 fresh specimens of lung cancer and paired normal lung tissue were obtained from 59 NSCLC cases treated in the department of thoracic surgery in our hospital from June 2006 to October 2007. Western blotting and immunohistochemistry were used to assay the specimens with goat anti-human EYA2 polyclone antibody. Clinicopathological parameters were collected and the correlation with EYA2 expression was subsequently analyzed., Results: The expression of EYA2 was detected in cytoplasm and nucleus of the cancer cells, but mostly in cytoplasm. Western blotting and immunohistochemistry showed the expression of EYA2 in NSCLC was increased and correlated with pathological type, but not with gender, age, pTNM stage, histological differentiation and lymph node metastasis. EYA2 expression was significantly up-regulated in adenocarcinoma, while not changed in lung squamous cell carcinoma., Conclusion: The results of this study suggest that expression of EYA2 in lung adenocarcinoma is augmented. EYA2 is likely participating in the development of lung adenocarcinoma as a transcriptional activator.

High mobility group box-1 protein, an abundant and conserved constituent of vertebrate nuclei, has recently been reported to be an endogenous immune signal [Rovere-Querini P, Capobianco A, Scaffidi P, Valentinis B, Catalanotti F, Giazzon M, et al. HMGB1 is an endogenous immune adjuvant released by necrotic cells. EMBO Reports 2004;5:825-30]. High mobility group box-1 protein can trigger the release of interleukin-2 and interleukin-12 from lymphocytes. However, at present the underlying mechanism remains unknown. It has been clarified that nuclear factor of activated T cells-2 transduces most immunological signals in T cells and modulates the production of interleukin-2. So it is natural that we asked whether high mobility group box-1 protein could promote production of interleukin-2 in a nuclear factor of activated T cells-2-dependent way. Our experiments firstly showed that high mobility group box-1 protein could bind to nuclear factor of activated T cells-2 in vivo and in vitro. High mobility group box-1 protein cotransfection markedly upregulated the transcription activity of nuclear factor of activated T cells-2 in promoting interleukin-2 reporter gene transcription, which was demonstrated to be dose-dependent. Cotransfection of high mobility group box-1 protein and nuclear factor of activated T cells-2 induced an 18.4-time increase of interleukin-2 activity in 293T cells and a 117.7-time increase in Hela cells. Moreover, inhibition of either high mobility group box-1 protein or nuclear factor of activated T cells -2 expression by sRNAi led to significant decrease of transcription activity of interleukin-2 reporter gene, suggesting that high mobility group box-1 protein and nuclear factor of activated T cells-2 both take important roles in facilitating interleukin-2 transcription, and high mobility group box-1 protein could act as a coactivator for nuclear factor of activated T cells-2 in enhancing transcription of interleukin-2. This discovery has not been reported elsewhere, and helps to understand the newly highlighted immunological role of high mobility group box-1 protein.

Nuclear factor of activated T cells 3 (NFAT3) activities have been implicated in many biological processes, such as breast cancer, cardiac hypertrophy, learning and memory, and adipocyte differentiation. However, how protein factors regulate NFAT3 transcriptional activity is poorly understood. Here, we report that regardless of estrogen, overexpression of estrogen receptor alpha and beta (ERalpha and ERbeta) suppresses NFAT3 transcriptional activity, whereas knockdown of endogenous ERalpha and ERbeta enhances the activity. Estrogen further enhances ER inhibition of NFAT3-dependent transcription. ERalpha and ERbeta interact with NFAT3 independently of the NFAT agonists phorbol myristate acetate (PMA) and ionomycin, and ERalpha is recruited to an NFAT3 target gene promoter. Phosphorylation of ERalpha at different sites differentially affects ERalpha modulation of NFAT3 transcriptional activity. These results suggest that ER may play a critical role in regulation of NFAT3 transcriptional activity.

Objectives: To study the effects of trichostatin A (TSA) on protein-protein interaction between HBx and histone deacetylase protein 1 (HDAC1)., Methods: Both HBx and HDAC1 expressing vectors were constructed by the method of routine molecular cloning. The expression of HBx and HDAC1 were observed by Western blot assay. The protein-protein interaction was tested between HBx and HDAC1 by GST pull-down in vitro as well as co-immunoprecipitation in vivo., Results: Both HBx and HDAC1 expressing vectors were successfully constructed. Protein-protein interaction between HBx and HDAC1 existed both in vitro and in vivo. TSA, an inhibitor of HDAC1, had no effect on the interaction between HBx and HDAC1., Conclusions: HBx interacts with HDAC1 in vivo and in vitro in a non- TSA dependent way.

Objective: To construct estrogen receptor alpha (ERalpha) trans-activation system., Methods: The full length ERalpha and its different function regions [(transcriptional activation function 1 (AF1), DNA binding domain (DBD), and transcriptional activation function 2 (AF2)] were amplified from pcDNA3-ERalpha by PCR and cloned into the pGAL vector. The expressions of the recombinant plasmids constructed were detected via immunoblotting. The 293T cells transfected with recombinant plasmids of full length ERalpha, its different function regions and empty vector were divided into 5 groups; each group was divided into 2 parts which were treated with or without estrogen (E(2)). The transcriptional activity of each group was detected in 293T cells after the recombinant plasmid was co-transfected with 0.2 microg of estrogen receptor element luciferase (ERE-LUC) and 0.1 microg of plasmid expressing beta-galactosidase and treated with or without 10 nmol/L E(2) for 24 hours., Results: The full length ERalpha and its different function regions were expressed in the 293T cells. Compared with the empty pGAL vector, the transcription activities of full length ERalpha, AF1, AF2 and DBD recombinant plasmids were raised about 20.44 +/- 1.01, 2.09 +/- 0.11, 8.09 +/- 0.30 and 1.05 +/- 0.09 fold, respectively, with the induction of E(2) after transfection in the 293T cells., Conclusion: The trans-activation system of ERalpha has been successfully established.

To study the relationship between the structure of dermatan sulfate (DS) derivatives and their anti-thrombotic activities, DS-derived oligosaccharides (with different structures and relative molecular weight (M(r))) were prepared, and the effects of the DS-derived oligosaccharides on the activities of heparin cofactor II (HCII), activated protein C (APC), blood platelet, and vascular endothelial cells were studied. The major disaccharides of DS and polysulfated dermatan sulfate (PSDS) were IdoA-1-->3-GalNAc-4-OSO(3) and IdoA-2OSO(3)-1-->3-GalNAc4, 6-diOSO(3), respectively. The results showed that the consequence of the thrombotic inhibitory effects of DS and its derivatives were as follows: PSDS>low molecular weight polysulfated dermatan sulfate (LPSDS)>DS. Both DS and PSDS inhibited platelet aggregation in the concentration-dependent manner, and the IC(50) value of DS and PSDS is 12.7+/-1.3 and 28.6+/-0.9 mg/mL, respectively. DS oligosaccharides (DSOSs) and PSDS oligosaccharides (PSDSOSs) both significantly inhibited P-selectin expression on platelet surface (P<0.01), while DSOSs have no different effect compared with PSDSOSs. DSOSs and PSDSOSs significantly enhanced the activity of HCII in inhibiting thrombin in the plasma. The most active PSDSOS was PSDSOS(1) with M(r) of 4959, which enhanced the HCII activity by 91% (P<0.01). The experiments on APC activity showed that DS and its derivatives enhanced APC activity. The most active PSDSOS was PSDSOS(3) with M(r) of 2749, which enhanced the APC activity to 331+/-27% (P<0.01). DSOSs and PSDSOSs enhanced tissue plasminogen activator (t-PA) activity and reduced the plasminogen activator inhibitor (PAI) activity from cultured human umbilical vein endothelial cells (HUVEC), resulting in the ratio of t-PA/PAI going up. PSDSOSs which have the same M(r) as DSOSs produced more active effects in above assays, except for platelet aggregation.

Aim: To prepare and characterize antibody against Memo protein and to detect the tissue distribution of Memo in mice., Methods: Fusion protein GST-Memo was expressed and purified, and polyclonal antibody against Memo was prepared by immunizing mice. A FLAG-tagged eukaryotic expression vector pcDNA3-FLAG-Memo was constructed. The specificity of the antibody was detected by Western blot., Results: An eukaryotic expression vector pcDNA3-FLAG-Memo was obtained. The polyclonal antibody was found to be specific to Memo. Memo protein was widely expressed in mouse tissues using the obtained antibody in Western blot., Conclusion: Antibody specific to Memo has been successfully obtained, which provides useful tool for investigation into Memo-associated mechanisms of tumor metastasis and invasiveness.

Objective: To investigate the features of HBx protein distributed in liver cells and its expression in E. coli., Methods: The expression vectors encoding the full length HBx and its mutants were constructed by the routine molecular cloning method. HBx protein expression was detected using Western blotting. The distribution feature of HBx protein in liver cells was examined using the fluorescence confocal microscopy. A series of purified HBx fusion proteins were obtained by glutathione-sepharose 4B affinity chromatography., Results: The expression vectors were successfully constructed for the full length HBx and its mutants. HBx was found distributed uniformly in the nuclei but granularly in the cytoplasm of the liver cells. Under optimal conditions, the mutant GST-HBx (72-120aa) was easily degraded., Conclusion: This study may provide a basis for further study on the biological function of HBx at the protein level.

Objective: To study the effects of exogenous ER beta on the growth of breast cancer MCF-7 cells under different treatment., Methods: An eukaryotic expression vector containing 1.6 kb of human entire coding sequence of ER beta (pCDNA3-ER beta) was transfected into human breast cancer MCF-7 cells using lipofectamine 2000. The biological activity of ER beta was detected with the luciferase reporter containing estrogen responsive element (ERE) and the expression of ER beta protein by Western blot. The growth properties of MCF-7, pCDNA 3-transfected MCF-7 and pCDNA 3-ER beta-transfected MCF-7 cells under different treatment, including E2 (17beta-estradiol) and 4-OHT (4-hydroxytamoxifen), were observed., Results: A stronger activation of the reporter by ER beta in the presence of E2 was observed in the pCDNA 3-ER beta-transfected MCF-7 cells than in the pCDNA 3-transfected MCF-7 and in MCF-7 cells. Western blot analysis showed that the protein level of ER beta in the pCDNA 3-ER beta-transfected MCF-7 cells was markedly increased. Exogenous ER beta expression did not change the growth properties and the morphology of MCF-7 cells under normal condition. The pCDNA 3-ER beta-transfected MCF-7 cells proliferated at the same rate as naive cells in the presence of 4-OHT, whereas a strong inhibition of the proliferation of the pCDNA 3-ER beta-transfected MCF-7 cells in the presence of E2 was observed., Conclusion: Exogenous ER beta expression does not increase the resistance to 4-OHT, and a strong inhibition of the proliferation may occur in the presence of E2.

Although eukaryotic translation initiation factor 5A (eIF5A) was originally designated as an "initiation factor," recent data have shown it to be also involved in apoptosis. However, the actual function of eIF5A in apoptosis is still unknown. In this study, we performed yeast two-hybrid screens to identify eIF5A-interacting proteins to help us understand the mechanisms of eIF5A. Our results demonstrated that eIF5A and syntenin could engage in a specific interaction both in vitro and in vivo and functioned collaboratively to regulate p53 activity. Our findings, for the first time, revealed a new biological activity for eIF5A as the regulator of p53. Overexpression of eIF5A or its EFP domain resulted in up-regulation of p53, and silencing eIF5A by small interfering RNA reduced the p53 protein level. Further analysis by reverse transcription PCR showed eIF5A-activated p53 transcription. The effect of eIF5A on p53 transcriptional activity was further demonstrated by the increasing expressions of p21 and Bax, well known target genes of p53. In contrast, a point mutant of eIF5A, hypusination being abolished, was revealed to be functionally defective in p53 up-regulation. Overexpression of eIF5A led to a p53-dependent apoptosis or sensitized cells to induction of apoptosis by chemotherapeutic agents. However, when eIF5A interacted with its novel partner, syntenin, the eIF5A-induced increase in p53 protein level was significantly inhibited. Therefore, eIF5A seems to be a previously unrecognized regulator of p53 that may define a new pathway for p53-dependent apoptosis, and syntenin might regulate p53 by balancing the regulation of eIF5A signaling to p53 for apoptosis.

Estrogen receptor alpha (ERalpha) signaling is paramount for normal mammary gland development and function and the repression of breast cancer. ERalpha function in gene regulation is mediated by a number of coactivators and corepressors, most of which are known to modify chromatin structure and/or influence the assembly of the regulatory complexes at the level of transcription initiation. Here we describe a novel mechanism of attenuating the ERalpha activity. We show that cofactor of BRCA1 (COBRA1), an integral subunit of the human negative elongation factor (NELF), directly binds to ERalpha and represses ERalpha-mediated transcription. Reduction of the endogenous NELF proteins in breast cancer cells using small interfering RNA results in elevated ERalpha-mediated transcription and enhanced cell proliferation. Chromatin immunoprecipitation reveals that recruitment of COBRA1 and the other NELF subunits to endogenous ERalpha-responsive promoters is greatly stimulated upon estrogen treatment. Interestingly, COBRA1 does not affect the estrogen-dependent assembly of transcription regulatory complexes at the ERalpha-regulated promoters. Rather, it causes RNA polymerase II (RNAPII) to pause at the promoter-proximal region, which is consistent with its in vitro biochemical activity. Therefore, our in vivo work defines the first corepressor of nuclear receptors that modulates ERalpha-dependent gene expression by stalling RNAPII. We suggest that this new level of regulation may be important to control the duration and magnitude of a rapid and reversible hormonal response.

Estrogen receptor alpha (ERalpha) has been a primary target of treatment as well as a prognostic indicator for breast cancer. The level of human X-box binding protein 1 (XBP-1) mRNA was related with that of ERalpha in breast tumors and was over-expressed in some breast tumors. These previous studies suggested that XBP-1 may interact with ERalpha. XBP-1 has two isoforms, XBP-1S and XBP-1U, as the result of unique splicing. GST pull-down assay showed that both XBP-1S and XBP-1U bound to ERalpha in vitro. The binding of XBP-1S to ERalpha was stronger than that of XBP-1U to ERalpha. Co-immunoprecipitation revealed that the binding was in a ligand-independent manner. XBP-1S and XBP-1U interacted with the region of ERalpha that contains a DNA-binding domain. The ERalpha-interacting regions on XBP-1S and XBP-1U have been mapped to two regions, the N-terminal basic region leucine zipper domain (bzip) and the C-terminal activation domain. These findings suggest that XBP-1S and XBP-1U may participate in ERalpha signaling pathway through the mediation of ERalpha.

BRCA1 is one of the most important breast cancer susceptibility genes. It plays key roles in DNA damage repair, cell cycle checkpoint regulation, gene transcription chromatin stability, and cell proliferation. In this paper, advance in basic researches on BRCA1 is reviewed, and the role of BRCA1 in cancer development and progression is discussed, that may facilitate potential clinical application of BRCA1.

Estrogen receptor (ERalpha) plays an important role in the development and progression of breast cancer. Several recent studies have demonstrated that expression of human X-box binding protein 1 (XBP-1) is associated with ERalpha status in breast tumors and overexpressed in a subset of breast tumors. XBP-1 has two splicing variants, which were designated as XBP-1S and XBP-1U, respectively. However, little is known about the expression pattern of XBP-1S and XBP-1U in breast cancer cells and about their roles in ERalpha signaling. In this study, the expression of two splicing forms of XBP-1 was detected in breast cancer cell lines with RT-PCR. Estrogen response element (ERE) -containing luciferase reporter assay was used to determine the effects of XBP-1S and XBP-1U on the transcription activity of ERalpha in MDA-MB-435 breast cancer cells. The result showed that both XBP-1S and XBP-1U enhanced the transcription activity of ERalpha in a hormone-independent and dose-dependent manner and the activity of of XBP-1S is higher than that of XBP-1U. Enhancement of ERE-containing luciferase reporter gene expression by XBP-1S and XBP-1U was dependent on ERalpha. These data suggest that XBP-1S and XBP-1U may play important roles in breast cancer growth and progression through ERalpha signaling.

BRCA1 (breast cancer susceptibility gene-1) plays important roles in DNA damage repair, cell checkpoint regulation, gene transcription, chromosome stability, and apoptosis. At the C-terminus of BRCA1 is the activation domain with a number of acidic amino acid residues that includes two tandem repeats of BRCT(BRCT1 and BRCT2). In this study, to identify proteins that interact with the BRCT2 domain of BRCA1, the standard yeast two-hybird screen was performed. FHL2 was isolated from a human ovary library, with the BRCT2 domain of BRCA1 as bait. Furthermore, the specific interaction of FHL2 with the BRCT2 domain of BRCA1, but not with the BRCT1 domain of BRCA1 and the BRCT domain of Rap1, was verified by yeast mating. To confirm the interaction between BRCA1 and FHL2 in vitro, the GST pull-down assay was performed, the coding sequences of BRCT1 and BRCT2 domains were fused in-frame with the coding region of GST in the pGEX-2T vector, generating the pGST-BRCT1 and pGST-BRCT2 recombinant plasmids the fusion proteins GST-BRCT1 and GST-BRCT2 were expressed in E. coli DH5 alpha. The purified fusion proteins were obtained by GST-Sepharose 4B affinity chromatography. The purified fusion proteins were incubated with in vitro translated 35S-methinine-labeled FHL2. Consistent with the two-hybird results, FHL2 could specifically bind to the BRCT2 domain, but not BRCT1 in vitro. To further assess the binding specificity of FHL2 to the BRCT2 domain of BRCA1 in vivo, pFLAG-FHL2 and pHABRCT1/pHA-BRCT2 recombinant plasmids were cotransfected into 293T cells. Then the coimmunoprecipitation assay were performed. The results also showed that FHL2 specifically interacted with the BRCT2 domain in vivo. Furthermore, the coimmunoprecipitation assay demonstrated that FHL2 could interact with endogenous BRCA1 in vivo. These findings lay solid foundations for study on the function of BRCA1 and FHL2 in cancer development and progression.

The estrogen receptor (ERalpha) is a member of a large superfamily of nuclear receptors that regulates the transcription of estrogen-responsive genes. Several recent studies have demonstrated that human X-box binding protein 1 (XBP-1) mRNA expression is associated with ERalpha status in breast tumors. More recently, two forms of XBP-1 were identified due to their unique splicing. The two splicing variants of XBP-1 were designated XBP-1S and XBP-1U, respectively. In this study, the coding sequences of XBP-1S and XBP-1U were cloned respectively into the expression vector pcDNA3 harboring FLAG epitope, generating the recombinant plasmids pcDNA3-FLAG-XBP-1S and pcDNA3-FLAG-XBP-1U. Western blot analysis showed that both XBP-1S and XBP-1U were expressed in mammalian cells. To determine the effects of XBP-1S and XBP-1U on the transcriptional activity of ERalpha, MDA-MB-231 breast cancer cells were cotransfected with the expression vectors for ERalpha and either pcDNA3-FLAG-XBP-1S or pcDNA3-FLAG-XBP-1U. The results indicated that XBP-1S and XBP-1U enhanced ERalpha-mediated transcriptional activities in a hormone-independent manner. GST pull-down assay showed that both XBP-1S and XBP-1U interacted with ERalpha. These data suggest that XBP-1S and XBP-1U may play an important role in breast cancer growth and progression through ERalpha signaling.

FHL2, a member of LIM-only protein family, plays an important role in transcription regulation, apoptosis, cancer development and progression. In this study, a mammalian transcription activation system was constructed by using DNA binding domain(DBD) of GAL4 and luciferase reporter gene with DBD binding sequence, and used for mapping of FHL2 transcription activation domain. First, the coding sequence of GAL4-DBD was inserted into expression vector pcDNA3, generating the pDBD recombinant plasmid, then the wild-type FHL2 and its mutants were fused in-frame with GAL4-DBD, resulting in expression vectors for wild-type FHL2 and its mutants. All of the recombinant plasmids were transfected into 293T cells. Western blot assay showed that all of the fusion proteins were expressed. The analysis of FHL2 transcription activation properties by using the GAL4-luciferase reporter gene indicated that wild-type FHL2 had activation activity in both 293T and MCF-7 cells. The deletion of the half LIM domain at the N-terminus severely impaired the capacity of FHL2 to stimulate transcription. The mutant lacking the LIM domain at the C-terminus was totally inactive, while the deletion of two LIM domains at the C-terminus partially recovered its ability to stimulate transcription. The deletion of the second LIM domain at C-terminus did not alter the activation capacity of FHL2. These results suggest that the last LIM domain at the C-terminus of FHL2 is critical for its transcription activation function, the second LIM domain at the C-terminus may be a negative regulation region, but this negative regulation depends on the last LIM domain. Mapping of transcription activation domain of FHL2 lays solid basis for further study on various FHL2 functions.

Objective: To construct an ERbeta expression vector and study its expression and function in different cancer cells., Methods: Standard PCR was used to amplify the full-length coding sequence of ERbeta. The amplified ERbeta gene was cloned into the eukaryotic expression vector pCDNA3, generating pCDNA3-ERbeta. The ERbeta expression was detected by Western blot and in vitro translation. The biological activity of ERbeta was detected by transfecting the pCDNA3-ERbeta into SV40-transformed embryonic kidney cell line 293T,breast cancer cell lines MDA-MB-435, MDA-MB-436, SKBR3, and prostate cancer cell line PC-3, with reporters containing estrogen response elements., Results: The recombinant plasmid pCDNA3-ERbeta was confirmed by restriction analysis to contain the ERbeta gene. The 63 000 ERbeta expression was shown by Western blot and further confirmed by in vitro translation. The ERbeta expression in different cancer cells was demonstrated to stimulate the expression of the reporters containing estrogen response elements, ERE and C3., Conclusion: ERbeta protein is successfully expressed and has biological activity, laying solid foundation for further study on its role in cancer cells.

Breast cancer susceptibility gene 1(BRCA1) plays an important role in breast cancer development and progression. BRCA1 encodes a 1863-amino acid protein with two BRCA1 C-terminal (BRCT) domains at its C-terminus, BRCT1 and BRCT2. Many cancer-predisposing mutations are located in the BRCT domains, which have been shown to induce chromatin unfolding by use of an approach that allows visualization of large-scale chromatin structure through lac repressor/lac operator recognition. To map the important region of BRCT domain (amino acid residues 1642-1736), six deletion mutant constructs were made. The chromatin structure assay showed that amino acid residues 1691-1721 are involved in the induction of chromatin unfolding. To further localize the critical amino acid residues, ten alanine scanning mutant constructs were made. The chromatin structure assay demonstrated that the 1707IAGGK1711 region is critical for the chromatin unfolding activity. Based on the mapped important region, Blast analysis identified a novel homologous protein. Mapping of the BRCT1 domain may aid in the presymptomatic risk assessment and provide a valuable tool for further study on the BRCT1 structure and function.

Mutations in breast cancer susceptibility gene 1(BRCA1) account for approximately 40%-50% of familial breast cancer cases and for more than 80% of inherited breast and ovarian cancer cases. Many cancer-predisposing mutations are located in the C-terminal region that functions as a transcriptional activation domain, but most of the mutations in the transactivation domain identified to date cannot be readily distinguished as either disease-associated mutations or benign polymorphisms. Because chromatin structure regulation is an early event in gene transcription control, the chromatin unfolding activities of different transactivation domain variants were compared with that of the wild-type transactivation domain by use of an approach that allows visualization of large-scale chromatin structure through lac repressor/lac operator recognition. To do this, different constructs of the transactivation domain were selected as follows: (a) the wild-type transactivation domain; (b) two polymorphisms (S1613G and M1652I); and (c) four cancer-predisposing mutations (A1708E, M1775R, W1837R and Y1853 term). All of the constructs were made by fusing in frame with lac repressor. Western blot analysis indicated that all of the fusion proteins were expressed in A03 1 cells, in which multiple copies of the lac operator were integrated to produce a heterochromatic region of the genome. The chromatin unfolding assay showed that, like the wild-type transactivation domain, two variants that represent benign polymorphisms did not induce chromatin unfolding or only induced subtle change. Contrary to the behaviors of the wild type and two benign variants, four cancer-predisposing mutations in the transactivation domain superactivate the chromatin unfolding. The results suggest that the chromatin unfolding assay can aid in the characterization of deterious mutations in the C-terminal transactivation domain of BRCA1 and may provide more reliable presymptomatic risk assessment.

With PCR technology and methods of DNA recombination in vitro, the fusion protein of GM-CSF with MCAF, the facto that has a directing effect on cells to a target, was generated by the construction of recombinant plasmids pMG 01, pMG 02 and pMG 03 with different nucleotide composition between its SD sequence and the initiation codon ATG under the control of lambdaP(R) P(L) promoter of vector pBV 220. Although no stable secondary structure exist in the translation initiation regions of all the recombinant plasmid constructed, the expression levels of the products with DH5 alpha (pMG 02) and DH5alpha (pMG 03) are much higher than that with DH5alpha (pMG01) which hardly expressed the product. The assay of Western blot indicated that the expressed product reacted with MCAF and GM-CSF antibodies, respectively. The assay of biological activities showed that the expressed product had apparent monocyte chemoattractant activity and supported the growth of the human GM-CSF-dependent cell line TF1, suggesting that the biological functions of MCAF and GM-CSF are compatible