Reporting of Quantitative Protein Electrophoresis in Australia and New Zealand: a Call for Standardisation

Background

The lack of guidelines on reporting standards for protein electrophoresis may have led to significant differences in reports from different laboratories.

Objective

To determine the extent of variation in reporting of protein electrophoresis results in Australia and New Zealand.

Method

Questionnaires were distributed to laboratories throughout Australia and New Zealand asking about protein electrophoresis practices and reporting.

Results

Extensive variation was found in the following reporting practices: (a) units for urine Bence Jones protein (BJP); (b) reporting absence of a paraprotein rather than a normal pattern; (c) numerical reporting of all protein fractions or only the paraprotein; (d) warning of possible inaccuracy in the serum immunoglobulin result of the paraprotein type; (e) co-migration of a paraprotein with a normal serum protein; (f) use of a confirmatory test when a known paraprotein is no longer detectable.

Conclusions

A working party should be established to make recommendations on the reporting of protein electrophoresis. Implementation of such recommendations should reduce both report variation between laboratories and the risk of misinterpretation of reports.     

Diet-induced obesity in rats leads to a decrease in sperm motility

Background  

Obesity is rapidly becoming a worldwide epidemic that affects children and adults. Some studies have shown a relationship between obesity and infertility, but until now it remains controversial. Thus, the aim of the present study was to investigate the effect of high-fat diet-induced obesity on male reproductive parameters.     

长春新碱诱导建立人结肠癌多药耐受性小鼠模型及MDR1和MRP1基因表达

目的建立人结肠癌多药耐受性动物模型并初步探索其耐药机制。方法结合体内外诱导方法建立人结肠癌多药耐受性动物模型,利用VCR和CTX的肿瘤抑制实验评价其MDR特性;利用real-time PCR和West-ern blotting等方法分析其P-gp/MDR1和MRP1基因和蛋白的表达。结果肿瘤抑制实验结果显示,MDR和敏感型结肠癌模型的肿瘤生长速度差异不显著,MDR结肠癌动物模型对于VCR和CTX的耐药性均有较大程度的提高;表达分析结果显示,人结肠癌MDR动物模型的P-gp/MDR1表达水平有较大提高,而MRP1表达没有显著变化。结论人结肠癌多药耐受性动物模型具有较好的多药耐受性,其多药耐受性表型主要是由于P-gp/MDR1过量表达所导致。     

Structural investigations of calcium binding and its role in activity and activation of outer membrane phospholipase A from Escherichia coli

Outer membrane phospholipase A (OMPLA) is an integral membrane enzyme that catalyses the hydrolysis of phospholipids. Enzymatic activity is regulated by reversible dimerisation and calcium-binding. We have investigated the role of calcium by X-ray crystallography. In monomeric OMPLA, one calcium ion binds between two external loops (L3L4 site) at 10 A from the active site. After dimerisation, a new calcium-binding site (catalytic site) is formed at the dimer interface in the active site of each molecule at 6 A from the L3L4 calcium site. The close spacing and the difference in calcium affinity of both sites suggests that the L3L4 site may function as a storage site for a calcium ion, which relocates to the catalytic site upon dimerisation. A sequence alignment demonstrates conservation of the catalytic calcium site but evolutionary variation of the L3L4 site. The residues in the dimer interface are conserved as well, suggesting that all outer membrane phospholipases require dimerisation and calcium in the catalytic site for activity. For this family of phospholipases, we have characterised a consensus sequence motif (YTQ-X(n)-G-X(2)-H-X-SNG) that contains conserved residues involved in dimerisation and catalysis.     

海藻酸微囊替代DMSO和FBS的STO细胞冷冻保存研究

目的:改进现有的细胞冷冻保存方法,建立一个不含二甲基亚砜(DMSO)和血清(FBS)的高效冷冻保存方法,为细胞治疗等临床实践提供优质细胞.方法:海藻酸微囊包埋鼠胚成纤维细胞(STO细胞)后用不含DMSO和FBS的冷冻保存液进行冷冻保存.设四个对照组:添加10％DMSO和20％FBS的组、仅添加10％DMSO的组、仅添加20％FBS、DMSO和FBS均不添加组.在冷冻前后对各实验组细胞用台盼兰染色,进行细胞计数,计算细胞存活率,同时利用溴乙锭的二聚物(EthD)、钙黄绿素-AM(Calcein-AM)进行染色观察细胞的形态,且进一步验证细胞存活率;解冻复苏后用MTT法评估细胞的增殖速度和生长活力.结果:冷冻保存30天后对各组的细胞数量、细胞存活率、细胞形态和解冻复苏后细胞的生长活力进行比较发现,海藻酸微囊包埋冷冻组的细胞数、细胞存活率、细胞形态和生长活力均与添加DMSO和FBS的组之间无显著性差异,而与其它三个对照组呈显著性差异.结论:使用海藻酸微囊替代DMSO和FBS保存STO细胞,能有效的维持细胞形态、数量、存活率,同时不影响细胞的生长活力,从而建立了一个不含DMSO和FBS的高效冷冻保存方法.     

Are hamstrings activated to counteract shear forces during isometric knee extension efforts in healthy subjects?

The hamstring muscles have the potential to counteract anterior shear forces at the knee joint by co-contracting during knee extension efforts. Such a muscle recruitment pattern might protect the anterior cruciate ligament (ACL) by reducing its strain. In this study we investigated to what extent co-activation of the knee flexors during extension efforts is compatible with the hypothesis that this co-activation serves to counteract anterior tibial shear forces during isometric knee extension efforts in healthy subjects. To this aim, it is investigated whether co-activation varies with the required knee extension moment, with the knee joint angle, and with the position of the external flexing force relative to the knee joint. With unaltered moment and muscle activation, distal positioning of the flexing force on the tibia causes higher resultant (muscular plus external) forward shear forces at the knee as compared to proximal positioning. In ten subjects, knee flexor and extensor EMG was measured during a quasi-isometric positioning task for a range (5-50 degrees) of knee flexion angles. It was found that the co-activation of the knee flexors increased with the extension moment, but this increase was less than proportional (p<0.001). The extension moment increased 2.7 to 3.4 times, whereas the activation of Biceps Femoris and Semitendinosus increased only a factor 1.3 to 2.0 (joint angle dependent). Furthermore, a strong increase in co-activation was seen near full extension of the knee joint. The position of the external extension load on the tibia did not affect the level of co-contraction. It is argued that these results do not suggest a recruitment pattern that is directed at reduction of anterior shear forces in the knee joint during sub-maximal isometric knee extension efforts in healthy subjects.     

Microtubules and cyclic amp in human leukocytes: on the order of things

We have shown previously that the β-adrenergic agonist isoproterenol (2μM) and the phosphodiesterase inhibitor isobutylmethylxanthine (1 mM) produce a much greater increase in cyclic AMP in human leukocytes that have been pretreated with colchicine (or with other agents that affect microtubule assembly) than in control leukocytes. The effects of colchicines were both time- and dose-dependant. These and other data suggested that the generation of cyclic AMP is normally restricted by an intact system of cytoplasmic microtubules. If so, then the same time and dose dependencies might apply to other colchicines-induced changes in leukocyte function. We have now assayed the distribution of concanavalin A (Con A)-receptor complexes on the leukocyte membrane, taking into account that leukocytes competent to assemble microtubules show a uniform distribution of surface- bound Con A whereas microtubule-deficient cells accumulate Con A in surface caps. We have found that the effect of colchicine on capping is also both time- and dose dependent, and that the dose-response relationships conform to those required to increase cyclic AMP levels. These findings provide further evidence that both colchicine-induced Con-A capping and colchicine- induced cyclic AMP generation depend upon the relaxation of constraints normally imposed by cytoplasmic microtubules upon the plasma membrane, which limit, respectively, lateral mobility of the lectin-receptor complexes, and expression of hormone-sensitive adenylate cyclase. Moreover, colchicine-induced Con-A cap formation is not affected even by very large changes in leukocyte cyclic AMP levels. Thus, elevated cyclic AMP levels do not appear to promote the dissolution of microtubules; rather, the dissolution of microtubules permits the generation of increased amounts of cyclic AMP.     

Molecular evolution of cytochrome c oxidase: rate variation among subunit VIa isoforms

Cytochrome c oxidase (COX) consists of 13 subunits, 3 encoded in the mitochondrial genome and 10 in the nucleus. Little is known of the role of the nuclear-encoded subunits, some of which exhibit tissue-specific isoforms. Subunit VIa is unique in having tissue-specific isoforms in all mammalian species examined. We examined relative evolutionary rates for the COX6A heart (H) and liver (L) isoform genes along the length of the molecule, specifically in relation to the tissue-specific function(s) of the two isoforms. Nonsynonymous (amino acid replacement) substitutions in the COX6AH gene occurred more frequently than in the ubiquitously expressed COX6AL gene. Maximum-parsimony analysis and sequence divergences from reconstructed ancestral sequences revealed that after the ancestral COX6A gene duplicated to yield the genes for the H and L isoforms, the sequences encoding the mitochondrial matrix region of the COX VIa protein experienced an elevated rate of nonsynonymous substitutions relative to synonymous substitutions. This is expected for relaxed selective constraints after gene duplication followed by purifying selection to preserve the replacements with tissue-specific functions.      

Determination of joint moments with instrumented force shoes in a variety of tasks

Ground reaction forces (GRFs) are often used in inverse dynamics analyses to determine joint loading. These GRFs are usually measured using force plates (FPs). As an alternative, instrumented force shoes (FSs) can be used, which have the advantage over FPs that they do not constrain foot placement. This study tested the FS system in one normal weight subject (77 kg) performing 19 different lifting, pushing and pulling and walking tasks. Kinematics were measured with an optoelectronic system and the GRFs and the positions of the centre of pressure (CoP) were synchronously measured with FPs and FSs. Differences between the outcomes of the two measurement systems (i.e. CoP and GRFs) and the resulting ankle and L5/S1 joint moments were determined at the instant of the peak GRF (DaPF). For most lifting and pushing and pulling tasks, the difference between the FP and FS measurements remained small: GRF DaPF remained below 3% body weight, CoP DaPF remained below 10 mm, ankle moment DaPF remained below 7% of the peak total ankle moment that occurred during normal walking and L5/S1 moment DaPF remained below 7% of the peak total L5/S1 moment that occurred during normal symmetric lifting. More substantial differences were only found in the maximal pushing tasks. For the walking tasks, peak vertical GRFs were somewhat underestimated. However, differences in ankle and L5/S1 moments remained small, i.e. DaPF below 7% of the peak total moment that occurred during normal walking.