Assessment of naturally occurring covalent and total dimer levels in human IgG1 and IgG2

Antibody dimers, two self-associated monomers, have been detected on both recombinantly expressed and endogenous human IgG proteins. Nearly 10 years ago, Yoo et al. (2003) described low levels of IgG2 covalent dimer, in human serum, but did not quantify the levels. Here we quantify the total and covalent dimer levels of IgG2 and IgG1 in human blood, and study the origin of covalent dimer formation. Low levels (<1%) of total IgG1 and IgG2 dimers were measured in freshly prepared human plasma. Both IgG1 and IgG2 covalent dimers were also found in plasma. Whereas IgG1 covalent dimer levels were significantly reduced by steps intended to eliminate artifacts during sample preparation, IgG2 covalent dimer levels remain stable in such conditions. About 0.4% of IgG2 in plasma was in a covalent dimer form, yet very little (<0.03%) of IgG1 covalent dimer could be considered naturally occurring. IgG2 dimer also formed in vitro under conditions designed to mimic those in blood, suggesting that formation occurs in vivo during circulation. Thus, small amounts of covalent IgG2 dimer do appear to form naturally.     

肿瘤坏死因子-α对泡沫细胞胆固醇流出及ATP结合盒转运子A1表达的影响

目的探讨肿瘤坏死因子-α（TNF-α）对泡沫细胞胆固醇流出途径的影响及可能作用机制.方法采用人组织瘤细胞-1（THP-1）细胞诱导分化形成的泡沫细胞,测定TNF-α与细胞胆固醇流出的时间、浓度关系.然后给予饱和浓度的TNF-α刺激,随机将泡沫细胞分成对照组、TNF-α组、对甲苯磺酰L-苯丙氨酸甲基甲酮（N-α-tosyl-L-phenylalanine chloromethy ketone,TPCK）组及TPCK＋TNF-α组,以RT-PCR法、Western blot法测定细胞ATP结合盒转运子A1（ABCA1）的表达.结果TNF-α抑制细胞胆固醇流出呈时间、浓度效应,在10.0 ng/ml时,TNF-α抑制胆固醇流出达到饱和效应.10.0 ng/ml TNF-α刺激后以时间依赖方式下调ABCA1表达.TPCK预孵育后可部分逆转TNF-α对ABCA1表达的下调.结论炎症因子TNF-α可通过抑制ABCA1的表达而减少细胞胆固醇的流出,核因子κB（NF-κB）激活抑制剂TPCK可逆转TNF-α的上述影响,提示TNF-α抑制ABCA1表达与NF-κB激活有关.TNF α/NF-κB信号途径可抑制泡沫细胞的胆固醇流出,从而加重细胞内胆固醇的蓄积。     

In the cut and thrust of apoptosis, serine proteases come of age

Proteolysis is central to the systematic cellular degradation that occurs during apoptosis. Predominantly, caspases have been studied in this regard. However, increasing evidence suggests that certain serine proteases may also play a significant role in apoptosis. Not only are these serine proteases involved in apoptosis signalling pathways independently, but they may also interact with more classical mediators of apoptosis such as the caspases or Bcl-2 family proteins. Isolation of apoptosis-associated serine proteases and the use of specific inhibitors have helped to shed light on potential pathways in which they are involved. Despite the recent developments in the field, knowledge regarding the role of serine proteases in apoptosis remains limited, but it is clear that investigations are gathering momentum and such studies may herald a new and exciting departure in apoptosis research.     

A novel modification of the Thrombelastograph assay, isolating platelet function, correlates with optical platelet aggregation

Flow cytometry, singlet platelet counting, and optical aggregation have been used to monitor clopidogrel and glycoprotein IIb/IIIa (GPIIb/IIIa) platelet antagonists. Optical aggregation is considered the gold standard, but neither it nor flow cytometry is convenient in larger-scale clinical studies or point-of-care systems. Singlet platelet counting, a point-of-care assay correlated with optical platelet aggregation, only provides a measurement of platelet function at a single point in time. The Thrombelastograph is used to assay whole blood for thrombin-generated maximal clot-shear elasticity, referred to as the maximal amplitude (MA). Although platelet dysfunction, thrombocytopenia, and the in vitro effect of strong inhibitors such as IIb/IIIa antagonists can be observed, with thrombin generation milder platelet inhibitors cannot be assessed. We modified the Thromboelastograph assay, using reptilase and factor XIIIa, to form a clot, without thrombin generation, in heparinized whole blood. The resulting clot MA is dependent on added platelet agonists such as ADP or arachidonic acid, is sensitive to platelet antagonists, and provides a continuous measure of platelet function more analogous and better correlated with optical aggregation. This novel modification of the Thromboelastograph assay should prove to be a useful point-of-care whole-blood assay with which to monitor the effects of GPIIb/IIIa, ADP, and thromboxane A(2)-receptor-inhibiting drugs in patients.     

Mitochondrial injury, oxidative stress, and antioxidant gene expression are induced by hepatitis C virus core protein

BACKGROUND & AIMS: The mechanisms of liver injury in chronic hepatitis C virus (HCV) infection are poorly understood. Indirect evidence suggests that oxidative stress and mitochondrial injury play a role. The aim of this study was to determine if the HCV core protein itself alters mitochondrial function and contributes to oxidative stress. METHODS: HCV core protein was expressed in 3 different cell lines, and reactive oxygen species (ROS) and lipid peroxidation products were measured. RESULTS: Core expression uniformly increased ROS. In 2 inducible expression systems, core protein also increased lipid peroxidation products and induced antioxidant gene expression as well. A mitochondrial electron transport inhibitor prevented the core-induced increase in ROS. A fraction of the expressed core protein localized to the mitochondria and was associated with redistribution of cytochrome c from mitochondrial to cytosolic fractions. Sensitivity to oxidative stress was also seen in HCV transgenic mice in which increased intrahepatic lipid peroxidation products occurred in response to carbon tetrachloride. CONCLUSIONS: Oxidative injury occurs as a direct result of HCV core protein expression both in vitro and in vivo and may involve a direct effect of core protein on mitochondria. These results provide new insight into the pathogenesis of hepatitis C and provide an experimental rationale for investigation of antioxidant therapy.     

Mechanism of action of 2-haloethylnitrosoureas on deoxyribonucleic acid. Nature of the intermediates from nitrosourea decomposition.

Direct current (DC) and differential pulse polarographic analyses were used to measure the rates of decomposition of a series of 2-haloethylnitrosoureas in aqueous solution. Measured by these methods, the rates of the first and rate-determining step which show a marked pH and solvent dependence agree with the overall rate of decomposition measured by gas evolution. In the 1,3-bis(haloethyl)-1-nitrosourea series, changing the nature of the halogen X has a small effect on the rate of decomposition. In the 3-cyclohexyl-1-(2-haloethyl)-1-nitrosourea series, changing X for OH or OCH₃ results in the rate of hydrolysis being reduced considerably. A free—NH₂ group in the nitrosourea structure as in CNU, MNU, ENU, CPNU, 4-CBNU and 5-CPNU accelerates considerably the rate of decomposition relative to the BCNU and CCNU series. Arrhenius parameters for the decomposition in aqueous pH 7.1 solution in the temperature range 28–47° were obtained for BFNU, BCNU and BBNU: log A, ?20.1± 1.4,?21.6± 0.7 and ?22.3±1.6; E_a, 24.4 ± 2.0, 26.5± 1.0 and 27.2 m 2.3 kcal/mole. The corresponding values for BINU were estimated as log A,?23.3± 3.0; E_a, 28.0± 3.0 kcal/mole. Examination of the decomposition products of 1,3-bis(2-chloropropyl)-1-nitrosourea (BCNU-β-Me) and 1,3-bisl 1-(chloromethyl)ethyl]-1-nitrosourea (BCNU-α-Me) favors decomposition pathway B via the diazohydroxide and cyclic chloronium ion for BCNU-β-Me and via the diazohydroxide and/or 2-chloro-1-methylethyl carbonium ion for BCNU-α-Me. While there is no evidence for the contribution of pathway A via a 2-imino-N-nitrosooxazolidinone for these compounds, consideration of product type and yields implicates a third decomposition pathway, via a 1,2,3-oxadiazoline intermediate. Additional evidence for an oxadiazoline intermediate is obtained by the isolation of 2-bromoethanol when BCNU is decomposed in the presence of a high concentration of sodium bromide.     

The Decanucleotide Polymorphism in the Factor VII Promoter Predicts Factor VII Plasma Levels but not the Risk of Acute Coronary Syndromes

Several preclinical studies have found a poor correlation between the ex vivo platelet inhibitory potency and the in vivo antithrombotic efficacy of GPIIb/IIIa receptor antagonists. The present study was designed to examine the differential in vitro potencies of c7E3, MK-383, DMP-728, and SM-20302 in inhibiting ex vivo platelet aggregation under normocalcemic and hypocalcemic conditions. Human blood was collected in either trisodium citrate (0.37%) or PPACK (20 µg/mL). Platelet aggregation assays were performed in platelet-rich plasma from citrate-anticoagulated blood (cPRP) and PPACK-anticoagulated blood (pPRP) using ADP (20 µM) and TRAP (10 µM) as agonists in the presence of c7E3, MK-383, DMP-728, or SM-20302. The concentration of ionized calcium in cPRP was 16–19 times lower than that in pPRP. The IC₅₀ of c7E3 for inhibiting ADP-induced platelet aggregation in cPRP (2.76 ± 0.11 µg/mL) was 1.6 times lower than that in pPRP (4.46 ± 0.48 µg/mL; P < 0.05). Similarly, the IC₅₀ for c7E3 for inhibiting TRAP-induced platelet aggregation in cPRP (4.52 ± 0.34 µg/mL) was 1.7 times lower than that in pPRP (7.69 ± 0.43 µg/mL; P < 0.05). MK-383, DMP-728, and SM-20302 also demonstrated 1.96-, 1.15-, and 1.43-fold lower IC₅₀ values, respectively, in cPRP as compared with pPRP. Chelation of ionized calcium in pPRP led to a progressive increase in platelet inhibition by all the antagonists. These results suggest that the observed in vitro inhibitory potency of a GPIIb/IIIa receptor antagonist is markedly enhanced when trisodium citrate is used as an anticoagulant to collect blood for ex vivo assay. These findings indicate that dosing regimens for GPIIb/IIIa receptor antagonists based on the platelet inhibition profile in citrate may provide misleading information with respect to their true in vivo antithrombotic efficacy.     

The novel pyrrolo-1,5-benzoxazepine, PBOX-21, potentiates the apoptotic efficacy of STI571 (imatinib mesylate) in human chronic myeloid leukaemia cells

The Bcr-Abl kinase inhibitor, STI571, is the first line treatment for chronic myeloid leukaemia (CML), but the recent emergence of STI571 resistance has led to the examination of combination therapies. In this report, we describe how a novel non-toxic G1-arresting compound, pyrrolo-1,5-benzoxazepine (PBOX)-21, potentiates the apoptotic ability of STI571 in Bcr-Abl-positive CML cells. Co-treatment of CML cells with PBOX-21 and STI571 induced more apoptosis than either drug alone in parental (K562S and LAMA84) and STI571-resistant cells lines (K562R). This potentiation of apoptosis was specific to Bcr-Abl-positive leukaemia cells with no effect observed on Bcr-Abl-negative HL-60 acute myeloid leukaemia cells. Apoptosis induced by PBOX-21/STI571 resulted in activation of caspase-8, cleavage of PARP and Bcl-2, upregulation of the pro-apoptotic protein Bim and a downregulation of Bcr-Abl. Repression of proteins involved in Bcr-Abl transformation, the anti-apoptotic proteins Mcl-1 and Bcl-_XL was also observed. The combined lack of an early change in mitochondrial membrane potential, release of cytochrome c and cleavage of pro-caspase-9 suggests that this pathway is not involved in the initiation of apoptosis by PBOX-21/STI571. Apoptosis was significantly reduced following pre-treatment with either the general caspase inhibitor Boc-FMK or the chymotrypsin-like serine protease inhibitor TPCK, but was completely abrogated following pre-treatment with a combination of these inhibitors. This demonstrates the important role for each of these protease families in this apoptotic pathway. In conclusion, our data highlights the potential of PBOX-21 in combination with STI571 as an effective therapy against CML.     

特戊酸氯甲酯合成新工艺的研究

目的 探讨合成特戊酸氯甲酯的简便工艺方法。方法 以己内酰胺作催化剂，特戊酸和氯化亚砜室温反应得到特戊酰氯，不经分离，补加适量氯化亚砜后直接与氯化锌和多聚甲醛室温反应合成特戊酸氯甲酯。结果 特戊酸氯甲酯总收率94．2％，产品结构经TR，1H NMR确认。结论 该法一步完成，条件温和，操作简单，值得进一步进行工业化开发。