Potential SARS-CoV-2 Mpro steroid inhibitors from Aphanamixis polystachya (Wall.) R. N. Parker.

Herein, six previously undescribed steroids (1-6), were isolated from leaves and twigs of Aphanamixis polystachya (Wall.) R. N. Parker (Meliaceae). Their structures were elucidated by comprehensive spectroscopic analysis, including HRESIMS, 1D and 2D NMR, UV, and IR. Antiviral activity of these compounds were evaluated. Compounds 1-6 showed varying degrees of inhibitory activity against the severe acute respiratory syndrome coronavirus 2 main protease (SARS-CoV-2 Mpro) at 200 µM.

Four new tirucallane-type triterpenoids from Aphanamixis polystachya.

Four new tirucallane-type triterpenoids, polystanins H-K (1-4), were obtained from the stems and leaves of Aphanamixis polystachya. Their structures were elucidated by analysis of the spectroscopic data and comparison with literature data. Compounds 1 and 2 showed week inhibitory effects against NO production in LPS-stimulated RAW264.7 cells. All the isolates were investigated for their antifungal activities against drug-resistant Candida albicans.

Triterpenoids from the fruits of Aphanamixis polystachya and their inhibitory activities on nitric oxide production.

Nineteen triterpenoids, including five previously unknown (four triucallane-type derivatives and one highly oxidized A, B-seco limonoids), together with fourteen known triterpenoids, were isolated from the fruits of Aphanamixis polystachya. Their structures were elucidated by extensive spectroscopic analysis. All isolates were evaluated their anti-inflammatory activities. The result showed that all compounds inhibit LPS-induced nitric oxide production in RAW264.7 macrophages with their IC50 value ranging from 95 to 1332 uM, and compound 6 exhibited obvious anti-inflammatory activity comparable to that of the positive control, with IC50 values of 94.96 uM.

Excretory/secretory products from Trichinella spiralis adult worms ameliorate myocardial infarction by inducing M2 macrophage polarization in a mouse model.

BACKGROUND: Ischemia-induced inflammatory response is the main pathological mechanism of myocardial infarction (MI)-caused heart tissue injury. It has been known that helminths and worm-derived proteins are capable of modulating host immune response to suppress excessive inflammation as a survival strategy. Excretory/secretory products from Trichinella spiralis adult worms (Ts-AES) have been shown to ameliorate inflammation-related diseases. In this study, Ts-AES were used to treat mice with MI to determine its therapeutic effect on reducing MI-induced heart inflammation and the immunological mechanism involved in the treatment. METHODS: The MI model was established by the ligation of the left anterior descending coronary artery, followed by the treatment of Ts-AES by intraperitoneal injection. The therapeutic effect of Ts-AES on MI was evaluated by measuring the heart/body weight ratio, cardiac systolic and diastolic functions, histopathological change in affected heart tissue and observing the 28-day survival rate. The effect of Ts-AES on mouse macrophage polarization was determined by stimulating mouse bone marrow macrophages in vitro with Ts-AES, and the macrophage phenotype was determined by flow cytometry. The protective effect of Ts-AES-regulated macrophage polarization on hypoxic cardiomyocytes was determined by in vitro co-culturing Ts-AES-induced mouse bone marrow macrophages with hypoxic cardiomyocytes and cardiomyocyte apoptosis determined by flow cytometry. RESULTS: We observed that treatment with Ts-AES significantly improved cardiac function and ventricular remodeling, reduced pathological damage and mortality in mice with MI, associated with decreased pro-inflammatory cytokine levels, increased regulatory cytokine expression and promoted macrophage polarization from M1 to M2 type in MI mice. Ts-AES-induced M2 macrophage polarization also reduced apoptosis of hypoxic cardiomyocytes in vitro. CONCLUSIONS: Our results demonstrate that Ts-AES ameliorates MI in mice by promoting the polarization of macrophages toward the M2 type. Ts-AES is a potential pharmaceutical agent for the treatment of MI and other inflammation-related diseases.

Two new triterpenoids from the fruits of Aphanamixis polystachya.

Two new triucallane triterpenoids, polystanin F (1) and polystanin G (2), along with eight known compounds (3-10) were isolated from the fruits of Aphanamixis polystachya. Their structures were established on the basis of extensive spectroscopic analysis. Moreover, eight compounds were evaluated for their in vitro cytotoxicity against three cancer cell lines (liver cancer RT112, colon cancer HCT-116 and breast cancer M231) using the MTT method. Compound 7 showed significant cytotoxic activity against HCT-116 with IC50 1.27 µM.

The Predictors of Conduction Disturbances Following Transcatheter Aortic Valve Replacement in Patients With Bicuspid Aortic Valve: A Multicenter Study.

Objective: To evaluate the predictors of new-onset conduction disturbances in bicuspid aortic valve patients using self-expanding valve and identify modifiable technical factors. Background: New-onset conduction disturbances (NOCDs), including complete left bundle branch block and high-grade atrioventricular block, remain the most common complication after transcatheter aortic valve replacement (TAVR). Methods: A total of 209 consecutive bicuspid patients who underwent self-expanding TAVR in 5 centers in China were enrolled from February 2016 to September 2020. The optimal cut-offs in this study were generated from receiver operator characteristic curve analyses. The infra-annular and coronal membranous septum (MS) length was measured in preoperative computed tomography. MSID was calculated by subtracting implantation depth measure on postoperative computed tomography from infra-annular MS or coronal MS length. Results: Forty-two (20.1%) patients developed complete left bundle branch block and 21 (10.0%) patients developed high-grade atrioventricular block after TAVR, while 61 (29.2%) patients developed NOCDs. Coronal MS <4.9 mm (OR: 3.08, 95% CI: 1.63-5.82, p = 0.001) or infra-annular MS <3.7 mm (OR: 2.18, 95% CI: 1.04-4.56, p = 0.038) and left ventricular outflow tract perimeter <66.8 mm (OR: 4.95 95% CI: 1.59-15.45, p = 0.006) were powerful predictors of NOCDs. The multivariate model including age >73 years (OR: 2.26, 95% CI: 1.17-4.36, p = 0.015), Δcoronal MSID <1.8 mm (OR: 7.87, 95% CI: 2.84-21.77, p < 0.001) and prosthesis oversizing ratio on left ventricular outflow tract >3.2% (OR: 3.42, 95% CI: 1.74-6.72, p < 0.001) showed best predictive value of NOCDs, with c-statistic = 0.768 (95% CI: 0.699-0.837, p < 0.001). The incidence of NOCDs was much lower (7.5 vs. 55.2%, p < 0.001) in patients without Δcoronal MSID <1.8 mm and prosthesis oversizing ratio on left ventricular outflow tract >3.2% compared with patients who had these two risk factors. Conclusion: The risk of NOCDs in bicuspid aortic stenosis patients could be evaluated based on MS length and prosthesis oversizing ratio. Implantation depth guided by MS length and reducing the oversizing ratio might be a feasible strategy for heavily calcified bicuspid patients with short MS.

Exploiting Joint-Resident Stem Cells by Exogenous SOX9 for Cartilage Regeneration for Therapy of Osteoarthritis.

The lack of effective treatment options for osteoarthritis (OA) is mostly due to the very limited regenerative capacity of articular cartilage. Mesenchymal stem cells (MSCs) have been most extensively explored for cell-based therapy to induce cartilage regeneration for OA. However, current in vitro expanded MSC-based approaches have significant drawbacks. On the other hand, osteoarthritic joints contain chondrocyte progenitors and MSCs in several niches which have the potential yet fail to differentiate into chondrocytes for cartilage regeneration. One of the underlying mechanisms of the failure is that these chondrocyte progenitors and MSCs in OA joints are deficient in the activity of chondrogenic transcription factor SOX9 (SRY-type high-mobility group box-9). Thereby, replenishing with exogenous SOX9 would reactivate the potential of these stem cells to differentiate into chondrocytes. Cell-permeable, super-positively charged SOX9 (scSOX9) protein is able to promote hyaline-like cartilage regeneration by inducing chondrogenic differentiation of bone marrow derived MSCs in vivo. This scSOX9 protein can be administered into osteoarthritic joints by intra-articular injection. This one-step, cell-free supplement of exogenous SOX9 may harness the regenerative potential of the intrinsic MSCs within the joint cavity to stimulate cartilage regeneration in OA.

Long-term durable repaired cartilage induced by SOX9 in situ with bone marrow-derived mesenchymal stem cells.

Background: Microfracture is a common procedure for cartilage repair, but it often produces inferior fibrocartilage. We previously reported that a super positively charged SOX9 (scSOX9) promoted hyaline-like cartilage regeneration by inducing bone marrow derived mesenchymal stem cell differentiation into chondrocytes in vivo. Here we examined the long-term efficacy of cartilage repair induced by microfracture with scSOX9 by assessing the biomechanical property of the repaired cartilage. Methods: A cartilage defect was created at the right femoral trochlear groove in New Zealand female rabbits and microfracture was performed. The scSOX9 protein was administered at the site of microfracture incorporated in a collagen membrane. Results: At 12 and 24 weeks, scSOX9 treatment induced hyaline-like cartilage while collagen-membrane alone induced fibrocartilage and mutant scSOX9-A76E poorly induced cartilage repair. The cartilage matrix in scSOX9-treated group showed highly enriched proteoglycan content. Consistent with the histological feature and the thickness of the repaired cartilage, the mechanical property of scSOX9-induced cartilage was also similar to that of normal cartilage. Conclusion: This long-term in vivo study demonstrated that in combination with microfracture, scSOX9 was able to induce reparative tissue with features of hyaline cartilage which was durable in long-term. This technology has the potential to translate into clinical use for cartilage repair to prevent progression to osteoarthritis.

Therapeutic efficacy of Schistosoma japonicum cystatin on sepsis-induced cardiomyopathy in a mouse model.

BACKGROUND: Myocardial dysfunction is one of the most common complications of multiple organ failure in septic shock and significantly increases mortality in patients with sepsis. Although many studies having confirmed that helminth-derived proteins have strong immunomodulatory functions and could treat inflammatory diseases, there is no report on the therapeutic effect of Schistosoma japonicum-produced cystatin (Sj-Cys) on sepsis-induced cardiac dysfunction. METHODS: A model of sepsis-induced myocardial injury was established by cecal ligation and puncture (CLP) in mice. Upon CLP operation, each mouse was intraperitoneally treated with 10 µg of recombinant Sj-Cys (rSj-Cys). Twelve hours after CLP, the systolic and diastolic functions of the left ventricular were examined by echocardiography. The levels of myoglobin (Mb), cardiac troponin I (cTnI), N-terminal pro-Brain Natriuretic peptide (NT-proBNP) in sera, and the activity of myeloperoxidase (MPO) in cardiac tissues were examined as biomarkers for heart injury. The heart tissue was collected for checking pathological changes, macrophages and pro-inflammatory cytokine levels. To address the signaling pathway involved in the anti-inflammatory effects of rSj-Cys, myeloid differentiation factor 88 (MyD88) was determined in heart tissue of mice with sepsis and LPS-stimulated H9C2 cardiomyocytes. In addition, the therapeutic effects of rSj-Cys on LPS-induced cardiomyocyte apoptosis were also detected. The levels of M1 biomarker iNOS and M2 biomarker Arg-1 were detected in heart tissue. The pro-inflammatory cytokines TNF-α and IL-6, and regulatory cytokines IL-10 and TGF-ß were measured in sera and their mRNA levels in heart tissue of rSj-Cys-treated mice. RESULTS: After rSj-Cys treatment, the sepsis-induced heart malfunction was largely improved. The inflammation and injury of heart tissue were significantly alleviated, characterized as significantly decreased infiltration of inflammatory cells in cardiac tissues and fiber swelling, reduced levels of Mb, cTnI and NT-proBNP in sera, and MPO activity in heart tissue. The therapeutic efficacy of rSj-Cys is associated with downregulated pro-inflammatory cytokines (TNF-α and IL-6) and upregulated regulatory inflammatory cytokines (IL-10 and TGF-ß), possibly through inhibiting the LPS-MyD88 signal pathway. CONCLUSIONS: RSj-Cys significantly reduced sepsis-induced cardiomyopathy and could be considered as a potential therapeutic agent for the prevention and treatment of sepsis associated cardiac dysfunction.

NLRP3 inflammasome expression in peripheral blood monocytes of coronary heart disease patients and its modulation by rosuvastatin.

Nucleotide­binding oligomerization domain, leucine rich repeat, and pyrin domain­containing protein 3 (NLRP3) inflammasome has been implicated in a series of physiological and pathological processes. However, its correlation in coronary heart disease (CHD) still remains to be elucidated. The present study aimed to determine the expression of NLRP3 inflammasome in peripheral blood monocytes (PBMCs) of stable angina pectoris (SAP) and acute myocardial infarction (AMI) patients. In addition, the effect of rosuvastatin on their activities was analyzed in vitro. A total of 60 participants with SAP (n=20), AMI (n=20) and non­CHD controls (n=20) were enrolled. Fluorescence­activated cell sorting, real­time PCR, western blotting and enzyme­linked immunosorbent assay were performed to reveal the role of NLRP3 inflammasome. NLRP3 inflammasome was expressed in the PBMCs, and revealed an increased expression along the downstream interleukin (IL)­1ß and IL­18 in both SAP and AMI groups, compared to the control group. Moreover, there was a more marked increase in the expression of these indicators in AMI patients when compared to SAP patients. Interference with rosuvastatin in vitro revealed that the expression of NLRP3 inflammasome and its downstream cytokines were significantly downregulated in both SAP and AMI groups in a time­dependent manner. The activation of NLRP3 inflammasome may be involved in the development of CHD, and rosuvastatin could attenuate the inflammatory process of atherosclerosis by downregulating NLRP3 expression and its downstream mediators. These findings indicated a potential role of NLRP3 in the pathogenesis and management of CHD, and also provided new insights into the mechanistic framework of rosuvastatin activity.

miR-93-3p alleviates lipopolysaccharide-induced inflammation and apoptosis in H9c2 cardiomyocytes by inhibiting toll-like receptor 4.

BACKGROUND: miR-93 is recently recognized to perform anti-inflammatory action in the pathological process of cardiomyocytes dysfunction. However, it remains unclear whether miR-93-3p involves in lipopolysaccharide (LPS)-induced inflammation and apoptosis in H9c2 cells. The present study aimed to investigate the functions of miR-93-3p and its target, toll-like receptor 4 (TLR4), in LPS-stimulated cardiomyocytes. MATERIAL AND METHODS: Cell viability was analyzed by CCK-8 assay. AnnexinV-FITC/PI staining and lactate dehydrogenase (LDH) assay were used to evaluate the cell death. The mRNA and protein levels were assayed by RT-qPCR and western blotting, respectively. The targeted gene was predicted by a bioinformatics algorithm and confirmed by a dual luciferase reporter assay. RESULTS: LDH stimulation resulted in the suppression of cell viability and the increase in apoptosis rate, inflammatory cytokines and LDH levels, while inhibition of TLR4 with TAK-242 or overexpression of miR-93-3p dramatically blocked LPS-induced inflammation and apoptosis in cardiomyocytes. Intriguingly, bioinformatics analysis and experimental data suggested that TLR4 was a direct target of miR-93-3p, which could inhibit TLR4 expression by transfected with miR-93-3p mimics or elevate the expression of TLR4 by transfected with miR-93-3p inhibitors. Overexpression of TLR4 carried out an opposite effect to miR-93-3p and positively regulated LPS-induced inflammation and apoptosis in cardiomyocytes. CONCLUSION: miR-93-3p showed the protective effects against LPS-induced inflammation and apoptosis in cardiomyocytes by inhibiting TLR4 expression.

MicroRNA­138 promotes proliferation and suppresses mitochondrial depolarization in human pulmonary artery smooth muscle cells through targeting TASK­1.

MicroRNA (miR)­138 serves an important role in the proliferation, differentiation and apoptosis of human pulmonary artery smooth muscle cells (HPASMCs), indi-cating the involvement of miR­138 in the development and progression of pulmonary artery hypertension (PAH). Potassium channel subfamily K member 3 (TASK­1), a two­pore domain K+ channel, is expressed in HPASMCs and is associated with hypoxic PAH. However, whether miR­138 mediates PAH through targeting TASK­1 is not known. In the present study, HPASMCs were transfected with miR­138 mimic to establish a PAH model in vitro, and the effects of a miR­138 inhibitor and a TASK­1 inhibitor (A293) were examined. Cell proliferation and mitochondrial membrane potential (MMP) were measured by CCK­8 assay and flow cytometry, respectively. Reverse transcription-quantitative polymerase chain reaction and western blotting were performed to examine the expression of miR­138, TASK­1, Bcl­2, caspase­3 and activation of extracellular signal­regulated kinase 1/2 (ERK1/2). A dual­luciferase reporter assay was also used to analyse the expression level of TASK­1 in HPASMCs. The results of the present study demonstrated that the miR­138 mimic promoted proliferation and MMP level, which was similar to the effect of A293 treatment on HPASMCs. However, the miR­138 inhibitor inhibited the effects induced by miR­138 mimic or A293 treatment, as demonstrated by a decrease in proliferation and MMP level in HPASMCs, accompanied by a decrease of Bcl­2 and an increase of caspase­3 expression levels, as well as ERK1/2 activation. The dual­luciferase reporter assay indicated that TASK­1 expression was negatively regulated by miR­138. The results of the present study suggested that miR­138 promoted proliferation and suppressed mitochondrial depolarization of HPASMCs by targeting TASK­1.

Regeneration of hyaline-like cartilage in situ with SOX9 stimulation of bone marrow-derived mesenchymal stem cells.

Microfracture, a common procedure for treatment of cartilage injury, induces fibrocartilage repair by recruiting bone marrow derived mesenchymal stem cells (MSC) to the site of cartilage injury. However, fibrocartilage is inferior biomechanically to hyaline cartilage. SRY-type high-mobility group box-9 (SOX9) is a master regulator of chondrogenesis by promoting proliferation and differentiation of MSC into chondrocytes. In this study we aimed to test the therapeutic potential of cell penetrating recombinant SOX9 protein in regeneration of hyaline cartilage in situ at the site of cartilage injury. We generated a recombinant SOX9 protein which was fused with super positively charged green fluorescence protein (GFP) (scSOX9) to facilitate cell penetration. scSOX9 was able to induce chondrogenesis of bone marrow derived MSC in vitro. In a rabbit cartilage injury model, scSOX9 in combination with microfracture significantly improved quality of repaired cartilage as shown by macroscopic appearance. Histological analysis revealed that the reparative tissue induced by microfracture with scSOX9 had features of hyaline cartilage; and collagen type II to type I ratio was similar to that in normal cartilage. This short term in vivo study demonstrated that when administered at the site of microfracture, scSOX9 was able to induce reparative tissue with features of hyaline cartilage.

Synthesis and Scanning Tunneling Microscopy Observation of 1,3,5-Triazine Bearing Azo-Carboxylate.

A new compound (2,4,6-tris-(3,5-di-methyl formate-4'-hydroxy azobenzene)-1,3,5-triazine,TDHAT) has been synthesized and the scanning tunneling microscopy (STM) is utilized to clarify the geometrical configuration on the highly oriented pyrolytic graphite (HOPG) surface. The star-shaped molecule self-assembles into uniform and regular triangular petal structure at liquid­solid interface, and high-resolution images has been obtained, which implied a high stability of this two dimensional configuration. The distance between the scattered bright spot and the center of the petal is measured to be about 1.2 nm (L2), which corresponds to the actual size of each arm. Moreover, a comparison has been made between the TDHAT molecule and the TMA 4a molecule which has similar star-shaped structure with a long alkyl chain at the each end of the three arms, drawing a conclusion that both the structure of arms and the substituent groups would impact the nanoarchitecture. The results give us insight into a better comprehension of the self-assembly of the star-shaped molecule, which benefits the construction of functional nanostructures.

Hydrogeochemical tracing of mineral water in Jingyu County, Northeast China.

The east Jilin Province in China, Jingyu County has been explored as a potential for enriching mineral water. In order to assess the water quality and quantity, it is of crucial importance to investigate the origin of the mineral water and its flow paths. In this study, eighteen mineral springs were sampled in May and September of 2012, May and September of 2013, and May 2014 and the environment, evolvement, and reaction mechanism of mineral water formation were analysed by hydrochemical data analysis, geochemical modelling and multivariate statistical analysis. The results showed that the investigated mineral water was rich in calcium, magnesium, potassium, sodium, bicarbonate, chloride, sulphate, fluoride, nitrate, total iron, silicate, and strontium, and mineral water ages ranged from 11.0 to more than 61.0 years. The U-shape contours of the mineral ages indicate a local and discrete recharge. The mineral compositions of the rocks were olivine, potassium feldspar, pyroxene, albite, and anorthite and were under-saturated in the mineral water. The origin of mineral water was from the hydrolysis of basalt minerals under a neutral to slightly alkaline and CO2-rich environment.

Identification of Risk Factors Affecting Impaired Fasting Glucose and Diabetes in Adult Patients from Northeast China.

BACKGROUND: Besides genetic factors, the occurrence of diabetes is influenced by lifestyles and environmental factors as well as trace elements in diet materials. Subjects with impaired fasting glucose (IFG) have an increased risk of developing diabetes mellitus (DM). This study aimed to explore risk factors affecting IFG and diabetes in patients from Northeast China. METHODS: A population-based, cross-sectional survey of chronic diseases and related risk factors was conducted in Jilin Province of Northeast China. All adult residents, aged 18-79, were invited to participate in this survey using the method of multistage stratified random cluster sampling. One hundred thirty-four patients with IFG or DM and 391 healthy control subjects were recruited. We compared demographic factors, body size measurements, healthy-related behaviors, and hair metallic element contents between IFG/diabetes patients and healthy individuals. RESULTS: IFG/diabetes patients had a greater weight, waist, hip, and body mass index (BMI) than control subjects. Significant differences in the content of zinc (Zn), potassium (K), copper (Ca), and sodium (Na) as well as Cu/Zn ratios between IFG or DM patients and control subjects (p < 0.05) were also observed. Hair Cu, selenium (Se), and Na contents were positively correlated with blood glucose levels (Cu: rs = 0.135, p = 0.002; Se: rs = 0.110, p = 0.012; Na: rs = 0.091, p = 0.038). Polytomous logistic regression adjusting for age, sex, family history of diabetes and BMI, showed that subjects with high BMI were more likely to develop IFG and DM (IFG: OR = 1.15, OR 95% CI = 1.02-1.29; DM: OR = 1.15, OR 95% CI = 1.01-1.33). Moreover, rarely or never eating fruits was a risk factor for DM (OR = 5.46, OR 95% CI = 1.87-15.98) but not for IFG (OR = 1.70, OR 95% CI = 0.72-4.02). Subjects with abdominal obesity or DM history were more susceptible to DM (abdominal obesity: OR = 2.99, OR 95% CI = 1.07-8.37; DM history: OR = 2.69, OR 95% CI = 1.01-7.20). We found that subjects living in Changling County had a significantly lower chance to suffer from IFG (OR and 95% CI for OR: 0.25, 0.08-0.74). Men and 60-69 years old subjects were at increased risk for IFG (male: OR = 3.51, OR 95% CI = 1.34-9.18; age 60-69: OR = 6.64, OR 95% CI = 1.36-32.47). We did not find significant associations of IFG or DM with certain lifestyles (such as eating more meat, exercise or physical activity, smoking, or alcohol drinking) or the content of some metallic elements (such as iron (Fe), Zn , K, calcium (Ca), Na, or magnesium (Mg)). CONCLUSIONS: This study demonstrated that less or no fruit eating, DM family history, abdominal obesity conferred vulnerability to DM. Living in Changling County, men and 60-69 years old subjects were found to be risk factors for IFG. Subjects with high BMI were more likely to develop IFG and DM.

Cell penetrating recombinant Foxp3 protein enhances Treg function and ameliorates arthritis.

Foxp3 is the master transcription factor for T regulatory (Treg) cell differentiation and function. This study aimed to test the therapeutic potential of cell penetrating recombinant Foxp3 protein in arthritis. Recombinant Foxp3 protein was fused to a cell penetrating polyarginine (Foxp3-11R) tag to facilitate intracellular transduction. In vitro Foxp3-11R treated CD4(+) T cells showed a 50% increase in suppressive function compared with control protein treated cells. Severity of arthritis in Foxp3-11R treated mice was significantly reduced compared with those treated with a control protein. CD4(+) T cells of lymph nodes and spleen from Foxp3-11R treated mice showed increased levels of Foxp3 expression compared with those of a control protein treated. These results demonstrated that Foxp3-11R can enhance T cell suppressive function and ameliorate experimental arthritis and suggest that cell penetrating recombinant Foxp3 is a potentially useful agent in therapy of arthritis.

Streptavidin suppresses T cell activation and inhibits IL-2 production and CD25 expression.

Streptavidin is widely used as a detection tool in biology research because of its high affinity and specificity binding to biotin. Biotin-streptavidin system has also been explored for detection of infection and tumor in clinical medicine. Here, we show immunosuppressive property of streptavidin on T cell activation and proliferation. Upon CD3 and CD28 stimulation, CD4(+) T cells produce interleukin 2 (IL-2) and express IL-2 receptor α chain (CD25). Addition of streptavidin in T cell culture suppressed IL-2 synthesis and CD25 expression with no cytotoxicity. The immunosuppressive effect of streptavidin was reversed by excessive biotin. Conjugated to a single chain anti-CD7 variable fragment (scFvCD7), streptavidin was directly delivered to T cells and showed substantially more profound suppressive effect on T cell activation. These results suggest that streptavidin could potentially be used as a novel immunomodulator.

Recombinant, refolded tetrameric p53 and gonadotropin-releasing hormone-p53 slow proliferation and induce apoptosis in p53-deficient cancer cells.

The p53 tumor suppressor is mutated in over 50% of human cancers. Mutations resulting in amino acid changes within p53 result in a loss of activity and consequent changes in expression of genes that regulate DNA repair and cell cycle progression. Replacement of p53 using protein therapy would restore p53 function in p53-deficient tumor cells, with a consequence of tumor cell death and tumor regression. p53 functions in a tetrameric form in vivo. Here, we refolded a wild-type, full-length p53 from inclusion bodies expressed in Escherichia coli as a stable tetramer. The tetrameric p53 binds to p53-specific DNA and, when transformed into a p53-deficient cancer cell line, induced apoptosis of the transformed cells. Next, using the same expression and refolding technology, we produced a stable tetramer of recombinant gonadotropin-releasing hormone-p53 fusion protein (GnRH-p53), which traverses the plasma membrane, slows proliferation, and induces apoptosis in p53-deficient, GnRH-receptor-expressing cancer cell lines. In addition, we showed a time-dependent binding and internalization of GnRH-p53 to a receptor-expressing cell line. We conclude that the GnRH-p53 fusion strategy may provide a basis for constructing an effective cancer therapeutic for patients with tumors in GnRH-receptor-positive tissue types.