Heterogeneous proteolytic specificity and activity of the house dust mite proteinase allergen Der p I

Background Exposure of the skin or respiratory tract to proteinases is frequently associated with allergic sensitization. This is of particular significance in the domestic indoor environment where the proteolytic activity of Der p I, the group I allergen of the house dust mite Dermatophagoides pteronyssinus, may influence the allergenicity of mites. Using class-specific proteinase inhibitors and active-site affinity chromatography, we have previously shown that Der p I exhibits a mixed cysteine-serine proteinase activity. Measurement of the amount of cleavage, however, did not determine whether the inhibitors used were targeting exactly the same proteolytic mechanism. Objective To resolve this issue, we have examined whether the cleavage specificity of the cysteine and serine proteinase activities of Der p I was the same. Methods HPLC and mass spectrometry were used to analyse and identify the products of a Der p I-digested peptide substrate and thus identify the peptide bonds cleaved. Results Der p I cleaves different peptide bonds, depending upon the class of proteolytic mechanism used. In the model peptide substrate insulin B chain, the cysteine and serine proteinase activities of Der p I showed preference for glutamic acid and arginine respectively in the P1 position. Conclusion These data suggest the existence of more than one mechanistic form of the allergen immunologically identified as Der p I. If proteolytic activity is indeed a function of allergenicity, this information may have important implications for the pathogenicity of Der p I and the ability of innate antiproteinase defences in the respiratory tract to prevent immune sensitization.     

智力低下者皮纹特征研究

通过对８３例智力低下者（男５８人，女２５人，ＩＱ２５～８９）的１７项指纹指标和７项掌纹指标特征的观察分析，并与７５２例中等智力者（男３７０人，女３８２人，ＩＱ９０～１０９）的指纹和掌纹进行比较。结果表明，两组间有１０项指标存在显著差异或高度差异，其中Ｗｃ、Ｗｂ、ｗＡｃ、Ｌ、Ｓｄ和Ｔｈ／１的真实花纹等６项指标与智力呈负相关；Ｗｓ、ｗｄｌ、Ｌｕ、ＰＴＣ等４项指标与智力呈正相关。同时，作者提出了Ｗｃ、Ｗｂ、ＷＡｃ、Ｓｄ和ＰＴＣ的正常值，其可作为判定智力低下的重要生物学指标，为筛查弱智儿童提供了一种简便易行的方法。作者还为皮纹学的研究方向提出了新的建议。同时，作者还发现了一种新的皮纹型，暂命名为Ｗｂ。     

Two-step hard acid deprotection/cleavage procedure for solid phase peptide synthesis

A new two-step deprotection/cleavage procedure for t-butoxycarbonyl (Boc) based solid phase peptide synthesis is reported. First the protective groups are removed from 4-(oxymethyl)-phenylacetamidomethyl (PAM) resin attached peptide with the weak hard acid, trimethylsilyl bromide-thioanisole/trifluoroacetic acid (TFA). In the second step, the peptide is cleaved from the resin with a stronger hard acid such as trimethylsilvl trifluoromethanesulfonate in TFA or with HF. The method is also shown to deformylate Nⁱⁿ-formyltryptophan moiety efficiently. The usefulness of this procedure for practical solid phase peptide synthesis is demonstrated by comparison with other deprotection methods in the synthesis of urotensin II and human endothelin.     

Granulosa cell metabolism and the assessment of oocyte quality in IVF

The progesterone production of the granulosa cells of the cumulus– oocyte complex correlates very well with the cleavagepotential of embryos in an IVF system. The method is simpleand can be easily performed by any laboratory associated withIVF. Furthermore, high intratubal progesterone levels in theimmediate post-ovulatory period are probably important in prolongingthe intra-ampullary residence of the oocyte or embryo untilthe uterine endometnum is optimal for implantation.     

New players in old amyloid precursor protein-processing pathways

The amyloid precursor protein (APP) gives rise to beta-amyloid peptides, which are the main constituents of senile plaques in brains of Alzheimer's disease (AD) patients. The generation of beta-amyloid peptides requires the enzymatic activity of the beta-site APP-cleaving enzyme 1 (BACE1). BACE1 is primarily expressed by neurons and increased BACE1 protein concentrations and enzymatic activities have been reported in the brains of AD patients. However, there is accumulating evidence that, in addition to neurons, reactive astrocytes are capable of expressing BACE1 and, therefore, may contribute to beta-amyloid plaque formation. This suggests that conditions accompanied by chronic astrocyte activation may contribute to developing AD. Non-amyloidogenic processing of the APP can be stimulated by phorbol esters (PEs) and by intracellular diacylglycerol (DAG) generation. This led to the hypothesis that classical and novel protein kinase Cs (PKCs), which are activated by DAG/PEs, regulate APP processing. However, in addition to PKCs, there are other DAG/PE receptors present in neurons which may participate in the modulation of APP processing. Munc13-1, a presynaptic protein with an essential role in synaptic vesicle priming, represents such an alternative target of the DAG second messenger pathway. Using Munc13-1 knock-out mice and human neuroblastoma cells transfected with wild-type and mutant Munc13-1 constructs it was demonstrated that Munc13-1 acts independently of and in parallel with PKC to modulate APP metabolism. Therefore, agonists specific for the Munc13-1 C1-domain or small molecules mimicking the function of the endogenous Munc13-1 activator RIM1 may prove useful to shift APP processing towards the non-amyloidogenic pathway.     

Effects of hammerhead ribozyme (RCP) on HBV P gene in vitro

Ｅｆｆｅｃｔｓｏｆｈａｍｍｅｒｈｅａｄｒｉｂｏｚｙｍｅ（ＲＣＰ）ｏｎＨＢＶＰｇｅｎｅｉｎｖｉｔｒｏＧａｎＬｉｘｉａ（甘立霞），ＷａｎｇＰｉｎｇ（王平），ＺｈｕＸｉｈｕａ（朱锡华），ＤｏｎｇＹａｎｌｉｎ（董燕麟），ＨｏｕＹｕｎｄｅ（侯云德）（Ｄｅｐａｒ...     

In vivo and in vitro studies of the site of inhibitory action of omeprazole on adrenocortical steroidogenesis

Summary The site of omeprazole inhibition of adrenal steroidogenesis has been sought in vivo by analyzing the patterns of urinary steroid metabolite excretion after 6 days of treatment with placebo/omeprazole.Excretion rates of androsterone, aetiocholanolone, dehydroepiandrosterone, 11 hydroxyandrosterone, tetrahydrocortisone, tetrahydrocortisol and cortolone were reduced, indicating a block at an early step in steroidogenesis, possibly cholesterol side-chain cleavage. In vitro studies have confirmed this finding by measuring conversion of added precursors to cortisol in isolated bovine adrenocortical cells. Cortisol synthesis from added 20 hydroxycholesterol was inhibited by 83% in the presence of 100 µg omeprazole/ml. Conversion from pregnenolone and progesterone and their 17 hydroxylated derivatives was inhibited by 20–40% whereas cortisol production from added 11 deoxycortisol was not affected.These data suggest that omeprazole primarily inhibits cholesterol cleavage and does not inhibit 3 hydroxysteroid dehydrogenase, 17 hydroxylase or 11 hydroxylation; 21 hydroxylase activity may be marginally attenuated.     

An integrative approach to CTL epitope prediction: a combined algorithm integrating MHC class I binding, TAP transport efficiency, and proteasomal cleavage predictions

Reverse immunogenetic approaches attempt to optimize the selection of candidate epitopes, and thus minimize the experimental effort needed to identify new epitopes. When predicting cytotoxic T cell epitopes, the main focus has been on the highly specific MHC class I binding event. Methods have also been developed for predicting the antigen-processing steps preceding MHC class I binding, including proteasomal cleavage and transporter associated with antigen processing (TAP) transport efficiency. Here, we use a dataset obtained from the SYFPEITHI database to show that a method integrating predictions of MHC class I binding affinity, TAP transport efficiency, and C-terminal proteasomal cleavage outperforms any of the individual methods. Using an independent evaluation dataset of HIV epitopes from the Los Alamos database, the validity of the integrated method is confirmed. The performance of the integrated method is found to be significantly higher than that of the two publicly available prediction methods BIMAS and SYFPEITHI. To identify 85% of the epitopes in the HIV dataset, 9% and 10% of all possible nonamers in the HIV proteins must be tested when using the BIMAS and SYFPEITHI methods, respectively, for the selection of candidate epitopes. This number is reduced to 7% when using the integrated method. In practical terms, this means that the experimental effort needed to identify an epitope in a hypothetical protein with 85% probability is reduced by 20-30% when using the integrated method.The method is available at http://www.cbs.dtu.dk/services/NetCTL. Supplementary material is available at http://www.cbs.dtu.dk/suppl/immunology/CTL.php.     

小鼠第一次卵裂周期中线粒体分布的变化

用线粒体专一性活体荧光染色剂罗丹明１２３显示小鼠受精卵在第一次卵裂周期中Ｍ的分布变化，雌原核和雄原核在汇合之前，Ｍ在细胞质中呈弥散状随机分布，两原核汇合后，Ｍ在核周围略显聚集，第一次卵裂后期，Ｍ沿纺锤体微管和２个子核周围集中，但在赤道区域内明显稀少，预示出细胞分裂面的定位，这说明细胞质分裂是收综环收缩和细胞结构调整共同作用的结果。２－细胞阶段，Ｍ在细胞核周围明显聚集。２－细胞胚受秋水仙素作用后，Ｍ     

Pregnancy outcome after blastocyst transfer as compared to early cleavage stage embryo transfer

BACKGROUND: Retrospective cohort study to evaluate differences in outcome when embryo transfer was performed either on day 2-3 (cleavage stage, CS-group) or on day 4-5 (blastocyst stage, BS-group). METHODS: A total of 1259 consecutive cycles yielding 500 live born babies performed at a single centre in Bregenz, Austria, were included. Main outcome measures were implantation and (multiple) pregnancy rates and neonatal outcome including birth defects. RESULTS: Total Pregnancy rate was 44% vs 28% (P < 0.001) and the total 'take home baby rate' was 37% vs 22% in the BS-group and the CS-group, respectively. Rate of multiple gestations (34% vs 17%, P = 0.001) was significantly higher among the BS-group, resulting in a higher rate of preterm deliveries < 36 weeks (26% vs 17%, P = 0.045). Female factor causing infertility (40% vs 21%, P < 0.001) was significantly higher among the BS-group. For the CS-group, rate of singleton pregnancies (83% vs 66%, P = 0.001) and idiopathic cause of infertility (34% vs 22%, P = 0.012) were significantly higher. No statistically significant differences were found in sex, Caesarean section rate, Apgar score and umbilical artery pH-values, total mean birth weight, admission rate to intensive care unit, days of hospitalization and number of minor and major birth defects. CONCLUSIONS: Our data suggest that blastocyst transfer may lead to a higher pregnancy rate with an overall better take-home baby rate (THBR) at the cost of higher rates of multiples and preterm deliveries.