A mono-sited transferrin from a representative deuterostome: the ascidian Pyura stolonifera (subphylum Urochordata)

An iron-binding protein has been found in the plasma of Pyura stolonifera. This protein has a molecular weight of about 41,000 +/- 2,000 and binds 1 mol iron/mol protein. The absorption maxima are lambda = 280 and lambda = 429 nm (E429/E280 = 0.044). Bicarbonate is bound concomitantly with high affinity and is necessary for optimal color formation at lambda = 429 nm. The protein showed a negligible exchange of iron with human apotransferrin under physiologic conditions over two hours. Upon incubation with rat reticulocytes, the protein reacts with membrane receptors for transferrins, and the protein, with its iron, is transported intracellularly where the iron is incorporated into heme. The 59Fe protein, after intravenous injection, disappears rapidly from the plasma and is excreted largely in the urine, with a substantial fraction present in the kidney and another large fraction present in the gut. These findings established the protein as a "transferrin" and support the concept that the larger transferrin molecule in vertebrates, with two iron-binding sites, resulted from a gene duplication.     

Further studies on proctolin analogues modified in position 2 of the peptide chain and their influence on heart-beat frequency of insects

Seven proctolin analogues (I-VII) modified in position 2 of the peptide chain by Phe (p-guanidino) (I), Phe (p-OEt) (II), Tyr (3′-NH₂) (III), Tyr (3′-NO₂) (IV), Afb (p-OH) (V) (Afb = 3-amino-4-phenyl-l -butyric acid), Afb (p-NH₂) (VI), Afb (p-NO₂) (VII), and the tetrapeptide Tyr (3′-NH₂)-Leu-Pro-Thr (VIII) were synthesized by the classic liquid-phase method. The biological effects of the peptides were investigated in cardioexcitatory tests on two insect species, the cockroach Periplaneta americana L., and the yellow mealworm, Tenebrio molitor L. Within physiological concentrations (10⁻⁹ - 10⁻⁷ M) peptides II, III, and IV stimulated the heart action of P. americana like proctolin itself. Under identical conditions, in the case of T. molitor, only peptide III showed cardiostimulatory properties, whereas other compounds (including II and IV) were inactive at concentrations up to 10⁻⁷m . Results reported here reflect, with reference to the analogues I-VII, selective recognition of receptors on myocardium of both insect species. The tetrapeptide VIII revealed a weak deacceleratory effect on P. americana and T. molitor heart action.     

Effects of phosphodiesterase type 5A inhibition on intracellular calcium handling and its implications for cardioprotection and antiarrhythmogenesis

Phosphodiesterase type 5A inhibition with sildenafil improves cardiac function in heart failure. In addition, sildenafil in animal models of myocardial infarction has direct cardioprotective and antiarrhythmic effects. Sildenafil reduces L-type calcium current (I_ca-L) and attenuates adrenergically driven inotropism, but effects on calcium handling are largely undetermined.Isolated adult rat ventricular myocytes were voltage clamped and calcium fluorescence measured with the indicator fura-2. Cells were paced at 0·5 Hz with depolarisations from ?60 mV to +10 mV. Sarcoplasmic reticulum (SR) content was determined by application of caffeine (10–20 mmol/L) and integration of inward sodium-calcium exchanger current. Rate constants for calcium extrusion from the cell (K_caff) and calcium uptake into the SR (K_SERCA) were determined by fitting first order exponentials to decay phases of the respective calcium transients. Following the initial control protocol, a therapeutically relevant dose of sidenafil (1 μM) was applied. Differences were determined with student's paired t tests.Sildenafil reduced SR content by 26·5% (n=9, p<0·01). To a lesser extent, sildenafil also reduced calcium transient amplitude (by ?13·6%, n=9, p<0·05); this was not accompanied by a reduction in K_SERCA (–2.3% with sildenafil, p=0·97, n=5). Peak and integrated I_ca-L were also reduced with sildenafil (–9·1% and ?6·0%, respectively, n=9, p<0·05). The effect on I_ca-L was also seen in adult dog ventricular myocytes (reducing peak and integrated I_ca-L by 15·9% and 26·4%, respectively, p<0·05 and p<0·01, n=6, 23°C). These effects cannot be attributed to run-down effects.Sildenafil substantially reduced SR content with no reduction in K_SERCA, and thus may be mediated through ryanodine receptor modulation. Such reduction in SR load may reduce proarrhythmic SR calcium release, indicating a novel mechanism through which sildenafil exerts an antiarrhythmic effect. Acute reductions in calcium transient amplitude and I_ca-L with sildenafil indicate acute negative inotropic effects and may contribute to our understanding of its cardioprotective effects in the setting of hyperadrenergic drive in heart failure.FundingBritish Heart Foundation.     

The C5a receptor antagonist PMX205 ameliorates experimentally induced colitis associated with increased IL-4 and IL-10

Background and Purpose

Anti-complement therapies have not been advanced for treating the inflammatory bowel diseases (IBDs) despite a growing body of evidence that blocking C5a protects against induced colitis in rodents. The purpose of this study was to further build on this evidence by examining the efficacy, mechanism and specificity of a potent, non-competitive and orally active C5a receptor (CD88) antagonist, PMX205, in the dextran sulphate sodium (DSS) model of murine innate colitis.

Experimental Approach

Mice with DSS added to their drinking water were orally administered 100 or 200 μg day⁻¹ PMX205 in prophylactic and therapeutic regimens. Clinical illness, colon histology and local generation of inflammatory mediators were measured to evaluate the impact of PMX205 on disease.

Key Results

PMX205 significantly prevented DSS-induced colon inflammation in both regimens, associated with lower pro-inflammatory cytokine production and nitrotyrosine staining in colon sections. Additionally, the levels of anti-inflammatory cytokines IL-4 and IL-10 were increased. PMX205 had no significant effect on C5a levels. The beneficial effect of PMX205 was seen in two strains of mice of differing sensitivities to DSS inflammation, but was inactive in mice lacking CD88.

Conclusions and Implications

Pharmacological inhibition of C5a activity by PMX205 is efficacious in preventing DSS-induced colitis, providing further evidence that targeting CD88 in IBD patients could be a valuable therapeutic option.     

Purification and characterization of heterogeneous pluripotent hematopoietic stem cell populations expressing high levels of c-kit receptor

Mouse pluripotent hematopoietic stem cells (PHSC) were fractionated based on size and density using counterflow centrifugal elutriation (CCE). These heterogeneous PHSC populations were further enriched by subtraction of cells with lineage-specific markers (Lin-) followed by positive sorting for c-kit expression. The cells were characterized for their functional and biochemical properties. We defined a subpopulation of c-kit-positive cells that expressed high numbers of c-kit receptors (c-kitBR). One hundred c-kitBR cells from either low- or higher-density fractions were sufficient to repopulate the lymphohematopoietic system in WBB6F1-W/Wv (W/Wv) recipients, whereas no PHSC were found in cells with low (c-kitDULL) or no (c-kitNEG) c-kit expression. Lin- c-kitBR cells were separated into RhoDULL and RhoBR subsets based on their ability to efflux rhodamine 123 (Rho). The PHSC were concentrated in Lin- c-kitBR RhoDULL cells and the number of Lin- c-kitBR RhoBR cells correlated directly with the number of day 12 colony-forming unit- spleen (CFU-S12) in each fraction. We were not able to enrich further for PHSC using monoclonal antibodies to the cell-surface markers AA4.1 or CD4, which have been used by others to isolate PHSC. The small, low- density Lin- c-kitBR subset contained PHSC and few CFU-S12. This enabled us to assay PHSC for expression of the flk-2 gene, which encodes a tyrosine kinase receptor present on fetal liver PHSC. Purified RNA from the low-density Lin- c-kitBR subset did not contain flk-2 mRNA. We suggest that AA4.1, CD4 and flk-2 are expressed as stage- specific markers on PHSC in cell cycle.     

主题综合疗法辅导戒毒患者的个案报告

目的:中国戒毒心理辅导工作起步较晚,还未形成有效的方法体系,本个案采用主题综合疗法对戒毒人员进行心理辅导,旨在探索有效的戒毒心理辅导模式。方法:心理辅导于2004-11/12在广州三九脑科医院进行,戒毒患者,男,25岁,吸毒史8年,采用主题综合疗法。根据戒毒人员普遍存在的心理问题和患者所存在的特殊问题,围绕培养积极生活态度,促进自我发展;增强戒毒信心,防止复吸的总目标,确定咨访关系建立;发现自我,建立戒毒信心;家庭关系及人际关系辅导;约定辅导4个主题,采用了半结构式的综合策略,贯穿着人本主义理念,以行为矫正法为主,综合了精神分析,认知疗法的具体技术。共进行了12次辅导,每次约50min,每周3次,总时程1个月。在心理辅导前后对患者进行药物依赖程度量表,抑郁自评量表和焦虑自评量表的测试,比较前后得分情况。结果:整个辅导获得了一定的成效,与患者建立了良好的咨询关系,在金钱管理、拒绝毒友、家庭沟通、共同约定几个主题效果较显著。患者主观上对海洛因依赖程度减轻,客观上药物依赖程度量表得分较辅导前降低(18,24);抑郁自评量表和焦虑自评量表得分均降低(抑郁量表前测粗分为40,后测为34,焦虑量表前测粗分为42,后测为32)。结论:主题综合戒毒疗法对于培养戒毒患者的积极生活态度,增强戒毒信心有明显效果,认知行为矫正技术是针对戒毒心理辅导的有效疗法。