The structural basis of the thermostability of SP1, a novel plant (Populus tremula) boiling stable protein

We previously reported on a new boiling stable protein isolated from aspen plants (Populus tremula), which we named SP1. SP1 is a stress-related protein with no significant sequence homology to other stress-related proteins. It is a 108-amino-acid hydrophilic polypeptide with a molecular mass of 12.4 kDa (Wang, W. X., Pelah, D., Alergand, T., Shoseyov, O., and Altman, A. (2002) Plant Physiol. 130, 865-875) and is found in an oligomeric form. Preliminary electron microscopy studies and matrix-assisted laser desorption ionization time-of-flight mass spectrometry experiments showed that SP1 is a dodecamer composed of two stacking hexamers. We performed a SDS-PAGE analysis, a differential scanning calorimetric study, and crystal structure determination to further characterize SP1. SDS-PAGE indicated a spontaneous assembly of SP1 to one stable oligomeric form, a dodecamer. Differential scanning calorimetric showed that SP1 has high thermostability i.e. Tm of 107 degrees C (at pH 7.8). The crystal structure of SP1 was initially determined to 2.4 A resolution by multi-wavelength anomalous dispersion method from a crystal belonging to the space group I422. The phases were extended to 1.8 A resolution using data from a different crystal form (P21). The final refined molecule includes 106 of the 108 residues and 132 water molecules (on average for each chain). The R-free is 20.1%. The crystal structure indicated that the SP1 molecule has a ferredoxin-like fold. Strong interactions between each two molecules create a stable dimer. Six dimers associate to form a ring-like-shaped dodecamer strongly resembling the particle visualized in the electron microscopy studies. No structural similarity was found between the crystal structure of SP1 and the crystal structure of other stress-related proteins such as small heat shock proteins, whose structure has been already determined. This structural study further supports our previous report that SP1 may represent a new family of stress-related proteins with high thermostability and oligomerization.     

Immunotherapy in high-risk chemotherapy-resistant patients with metastatic solid tumors and hematological malignancies using intentionally mismatched donor lymphocytes activated with rIL-2: a phase I study

The feasibility and safety of immunotherapy mediated by intentionally mismatched rIL-2 activated killer lymphocytes (IMAK) with no prior stem cell engraftment was investigated in patients with advanced chemotherapy-resistant hematological malignancies and metastatic solid tumors. Our goals were to maximize anti-cancer activity by using intentionally mismatched donor lymphocytes; amplify killing of target cancer cells by rIL-2 activation of killer cells in vitro and in vivo, and avoid the risk of graft-versus-host disease (GVHD) by anticipated rejection of alloreactive donor lymphocytes. Conditioning consisted of 5 days of fludarabine 25 mg/m² or a single dose of cyclophosphamide 1,000 mg/m², 2 subcutaneous injections of alpha interferon (IFN) 3 × 10⁶ and COX2 inhibitors, followed by administration of IMAK (65 ± 5 CD3⁺CD56⁻; 17 ± 5 CD3⁻CD56⁺) in conjunction with low dose subcutaneous rIL-2 (6 × 10⁶ IU/m²/day) for 5 days for continuous activation of alloreactive donor lymphocytes prior to their anticipated rejection. Here, we present our phase 1 clinical study data in a cohort of 40 high-risk patients with metastatic solid tumors and hematological malignancies. Treatment was accompanied by some malaise and occasional self-limited fever but otherwise well tolerated on an outpatient basis. Transient engraftment of donor cells was documented in two patients and only one developed self-limited grade 1 GVHD. Among patients with chemotherapy-resistant disease, long-term progression-free survival was recorded in 5 of 21 evaluable patients with metastatic solid tumors and in four of five patients with hematological malignancies. We conclude that the proposed procedure is feasible, safe, and potentially effective, with some otherwise resistant cancer patients long-term disease-free, thus justifying larger Phase II studies in patients with hematological malignancies and metastatic solid tumors, preferably at a stage of minimal residual disease with the goal in mind to eradicate all malignant cells at an early stage of the disease.     

The rising incidence of type 1 diabetes in south-eastern Poland. A study of the 0-29 year-old age group, 1980-1999

INTRODUCTION: Studies carried out over the last few years have provided information about the increase in the incidence of type 1 diabetes in different parts of the world including the European countries bordering Poland. THE AIM OF STUDY: The aim of this study was to determine the long-term trends in the incidence of type 1 diabetes over the 20 years between 1980 and 1999 and to compare the incidence during the decades preceding and following the 1989 economic and political transformation in Poland. MATERIAL AND METHODS: The registration of type 1 diabetes among people aged 0-29 was drawn up according to the DERI recommendations using three data sources. We calculated the age-standardised incidence rates for five-year age groups and determined the long-term trend in the incidence of type 1 diabetes in south-eastern Poland. RESULTS: A significant growth in the incidence of type 1 diabetes was observed among people aged 0-29 in the Rzeszów Province in the period between 1980 and 1999. The mean age-standardised incidence rate was 6.1/100,000, and a statistically significant difference was noted between the 1980s and the 1990s (5.3 [95%CI 4.5-6.0] and 6.8 [95%CI 5.9-7.6]). The male incidence of 6.7 significantly exceeded that for females--5.5/100,000. There was also a higher incidence in the group aged 0-14 in comparison with the group aged 15-29 (6.4 and 5.8/100,000 respectively). The highest incidence was found in boys aged 10-14 (11.5/100,000) and a significantly rising trend was observed in children of 0-4 years old. CONCLUSIONS: The mean incidence of type 1 diabetes among the study population was low. Nevertheless, we demonstrated a significantly increasing trend in the incidence during the 20-year observation period. The incidence in the 1990s, both in general and for males, was significantly higher when compared to the 1980s.     

Interleukin 2 regulation following semi-allogeneic bone marrow transplantation in mice

Success of allogeneic and autologous bone marrow transplantation (BMT) is hampered by susceptibility to infection during the first two post-treatment years. Further, in treating malignant diseases, impaired anti-host reactivity for donor cells may contribute to a high rate of relapse. Both complications are a consequence of immune deficiency involving B and T lymphocytes. The present study evaluates several key parameters of the immunologic reconstitution mechanism in mice subjected to myeloablative total body irradiation following semi-allogeneic (parental) BMT. This resulted in a gradual reduction of splenic CD3, CD4 and CD8 cells until day 45 post-BMT. Concomitantly, there was an increase in monocytes and CD4⁺/CD8⁺ (double positive) cells, accompanied by a persistent elevation in the percentage of B lymphocytes. The total thymic and splenic T cell populations were reduced until day +30. The cellular reduction correlated with the poor proliferative response of the thymic and splenic cells. A decrease occurred in IL-2 mRNA expression in thymic cells during days 15–20 post-transplant, corresponding with the low level of IL-2 secretion in the spleen and thymus of the transplanted mice. In conclusion, following semi-allogeneic BMT, there was an overall immune down-regulation in the cells, gene and protein levels. Reduced immunological responsiveness following BMT reinforces the need for improving the immune dysfunction by immunotherapy post-BMT.     

Synthesis and antibacterial activity of novel 6-O-substituted erythromycin A derivatives

A series of novel 6-O-substituted erythromycin A derivatives has been synthesized. Good in vitro antibacterial activity has been demonstrated for analogues incorporating a variety of structural features. The methodology disclosed is expected to find application in the design of future macrolide antibiotics that target the prevalent bacterial resistance problem.     

Molecular docking and enzyme kinetic studies of dihydrotanshinone on metabolism of a model CYP2D6 probe substrate in human liver microsomes

The effects of Danshen and its active components (tanshinone I, tanshinone IIA, dihydrotanshinone and cryptotanshinone) on CYP2D6 activity was investigated by measuring the metabolism of a model CYP2D6 probe substrate, dextromethorphan to dextrorphan in human pooled liver microsomes. The ethanolic extract of crude Danshen (6.25-100 μg/ml) decreased dextromethorphan O-demethylation in vitro (IC(50)=23.3 μg/ml) and the water extract of crude Danshen (0.0625-1 mg/ml) showed no inhibition. A commercially available Danshen pill (31.25-500 μg/ml) also decreased CYP2D6 activity (IC(50)=265.8 μg/ml). Among the tanshinones, only dihydrotanshinone significantly inhibited CYP2D6 activity (IC(50)=35.4 μM), compared to quinidine, a specific CYP2D6 inhibitor (IC(50)=0.9 μM). Crytotanshinone, tanshinone I and tanshinone IIA produced weak inhibition, with IC(20) of 40.8 μM, 16.5 μM and 61.4 μM, respectively. Water soluble components such as salvianolic acid B and danshensu did not affect CYP2D6-mediated metabolism. Enzyme kinetics studies showed that inhibition of CYP2D6 activity by the ethanolic extract of crude Danshen and dihydrotanshinone was concentration-dependent, with K(i) values of 4.23 μg/ml and 2.53 μM, respectively, compared to quinidine, K(i)=0.41 μM. Molecular docking study confirmed that dihydrotanshinone and tanshinone I interacted with the Phe120 amino acid residue in the active cavity of CYP2D6 through Pi-Pi interaction, but did not interact with Glu216 and Asp301, the key residues for substrate binding. The logarithm of free binding energy of dihydrotanshinone (-7.6 kcal/mol) to Phe120 was comparable to quinidine (-7.0 kcal/mol) but greater than tanshinone I (-5.4 kcal/mol), indicating dihydrotanshinone has similar affinity to quinidine in binding to the catalytic site on CYP2D6.     

Polysaccharide peptides from Coriolus versicolor competitively inhibit model cytochrome P450 enzyme probe substrates metabolism in human liver microsomes

Polysaccharide peptide (PSP), isolated from COV-1 strain of Coriolus versicolor, is commonly used as an adjunct in cancer chemotherapy or health supplement in China. Previous studies have shown that PSP decreased antipyrine clearance and inhibited rat CYP2C11-mediated tolbutamide 4-hydroxylation and in human CYP2C9. In this study, the effects of the water extractable fraction of PSP on the metabolism of model CYP1A2, CYP2D6, CYP2E1 and CYP3A4 probe substrates were investigated in pooled human liver microsomes. PSP (1.25-20μM) dose-dependently decreased CYP1A2-mediated metabolism of phenacetin to paracetamol (IC(50) 19.7μM) and CYP3A4-mediated metabolism of testosterone to 6β-hydroxytestosterone (IC(20) 7.06μM). Enzyme kinetics studies showed the inhibition of CYP1A2 activity was competitive and concentration-dependent (K(i)=18.4μM). Inhibition of testosterone to 6β-hydroxytestosterone was also competitive and concentration-dependent (K(i)=31.8μM). Metabolism of dextromethorphan to dextrorphan (CYP2D6-mediated) and chlorzoxazone to 6-hydroxychlorzoxazone (CYP2E1-mediated) was only minimally inhibited by PSP, with IC(20) values at 15.6μM and 11.9μM, respectively. This study demonstrated that PSP competitively inhibited the CYP1A2- and CYP3A4-mediated metabolism of model probe substrates in human liver microsomes in vitro. The relatively high K(i) values for CYP1A2 and CYP3A4 would suggest a low potential for PSP to cause herb-drug interaction related to these CYP isoforms.     

Enzyme kinetic and molecular docking studies on the metabolic interactions of 1-hydroxy-2,3,5-trimethoxy-xanthone, isolated from Halenia elliptica D. Don, with model probe substrates of human cytochrome P450 enzymes

Halenia elliptica D. Don is a Tibetan herb and medicinal preparations containing Halenia elliptica have been commonly used for the treatment of hepatitis B virus infection in China. The metabolism of 1-hydroxy-2,3,5-trimethoxy-xanthone (HM-1) to its metabolites is mediated through cytochrome P450 enzymes. This study aimed to investigate the herb-drug interaction potential of HM-1 by studying its effects on the metabolism of model probe substrates of five major CYP450 isoforms in human liver microsomes. HM-1 showed moderate inhibitory effects on CYP1A2 (IC(50)=1.06μM) and CYP2C9 (IC(50)=3.89μM), minimal inhibition on CYP3A4 (IC(20)=11.94μM), but no inhibition on model CYP2D6 (dextromethorphan) and CYP2E1 (chlorzoxazone) probe substrates. Inhibition kinetic studies showed that the K(i) values of HM-1 on CYP1A2, CYP2C9 and CYP3A4 were 5.12μM, 2.00μM and 95.03μM, respectively. HM-1 competitively inhibited testosterone 6β-hydroxylation (CYP3A4) but displayed mixed type inhibitions for phenacetin O-deethylation (CYP1A2) and tolbutamide 4-hydroxylation (CYP2C9). Molecular docking study confirmed the inhibition modes of HM-1 on these human CYP isoforms.