T. William Jordan

Fuhrman grading is not appropriate for chromophobe renal cell carcinoma.

This study was undertaken to assess the prognostic effectiveness of Fuhrman nuclear grading and the individual components of this grading system, in a series of chromophobe renal cell carcinomas. Eighty-seven cases of chromophobe renal cell carcinoma were investigated. There were 47 males and 40 females, 28 to 78 years of age. The carcinomas ranged from 25 to 180 mm in size and on TNM staging there were 38 stage I, 25 stage II, 22 stage III, and 2 stage IV tumors. Whole tumor Fuhrman grading was grade 1, 6 cases; grade 2, 72 cases; grade 3, 8 cases; and grade 4, 1 case, whereas focal (single high power field) grading was grade 1, 1 case; grade 2, 62 cases; grade 3, 21 cases; and grade 4, 3 cases. On assignment of nucleolar grading using Fuhrman criteria there were 37 grade 1, 44 grade 2, and 4 grade 3 tumors on whole tumor assessment and 3 grade 1, 63 grade 2, and 21 grade 3 tumors on assessment of the high power field showing the greatest degree of nuclear pleomorphism. Measurements of nuclear size showed nuclear area to range from 26.14 to 100.74 μm2, nuclear perimeter from 19.73 to 39.28 μm, and nuclear major axis from 6.49 to 13.21 μm, whereas the ranges of measures of nuclear shape were; shape factor 0.798 to 0.890, compactness 14.260 to 15.843, and feret diameter 5.694 to 11.242. Follow-up ranged from 1 to 150 months and 8 patients died of tumor-related causes 5 to 53 months from diagnosis. On log rank testing against survival, only patient age (P=0.016) and tumor maximum diameter (P=0.0055) were significant, whereas patient sex and TNM stage were not significant. Whole tumor and focal Fuhrman grading, as well as all measures of nucleolar prominence, nuclear size, and nuclear shape showed no significant association with outcome. It is concluded that neither Fuhrman grading, nor any of the components of the Fuhrman grading system, is useful as prognostic indicators for this tumor type.

Nucleolar grade but not Fuhrman grade is applicable to papillary renal cell carcinoma.

This study was undertaken to determine the validity of Fuhrman grading in a series of papillary renal cell carcinomas (PRCCs), to examine the interrelationship and prognostic significance of the individual components of the grading system, and further to determine whether any observed predictive value was independent of other prognostic indicators. Ninety cases of PRCC were studied. Fifty-nine tumors were of type 1 and 31 were of type 2. There were 33 TNM stage 1, 26 stage 2, 18 stage 3, and 12 stage 4 tumors, whereas division of cases according to pT category showed 14 pT1a, 20 pT1b, 25 pT2, 15 pT3a, 4 pT3b, and 11 pT4 tumors. Ten tumors were grade 1, 58 grade 2, and 22 grade 3 when predominant Fuhrman grade was assigned, whereas grading according to the high-power field containing the highest grade (focal grade) showed 40 grade 2, 49 grade 3, and 1 grade 4 tumors. Measurements of nuclear size (area, major axis, perimeter) and shape (shape factor, compactness) were undertaken using image analysis. Nuclear area ranged from 27.63 to 116.39 μM2, major axis length 6.70 to 14.06 μM, and nuclear perimeter 20.05 to 41.77 μM. Shape factor ranged from 0.805 to 0.878 and compactness from 14.33 to 15.66. Predominant nucleolar grade using the criteria of the Fuhrman classification was nucleolar grade 1 for 13 tumors, nucleolar grade 2 for 56 tumors, and nucleolar grade 3 for 21 tumors. Focal nucleolar grade based on the high-power field showing the greatest degree of nuclear pleomorphism, was grade 2 for 38 tumors and grade 3 for 52 tumors. pT category, TNM stage, focal Fuhrman grade, and PRCC type were significantly associated with survival. Of the various measures of the components of the Fuhrman classification, only focal nucleolar grade was associated with survival, on univariate analysis. On multivariate analysis, focal nucleolar grade and tumor diameter were independently associated with survival, whereas TNM stage retained significance independent of other parameters. It is concluded that assessment of nucleolar prominence rather than Fuhrman grade is applicable for stratification of tumors within TNM stage or pT category for PRCC and that this should be based upon the high-power field showing the greatest degree of nuclear pleomorphism.

Characterization of non-covalent oligomers of proteins treated with hypochlorous acid

Hypochlorous acid (HOCl) is a potent oxidant produced by myeloperoxidase that causes aggregation of many proteins. Treatment of apohaemoglobin and apomyoglobin with HOCl produced a regular series of oligomer bands when the proteins were separated by SDS/PAGE under reducing conditions. Aggregation was detectable at a HOCl/protein molar ratio of 0.5:1 and was maximal at ratios of 10:1-20:1. Dimers formed within 1 min of adding HOCl, and further aggregation occurred over the next 30 min. No convincing evidence for covalent cross-linking was obtained by amino acid analysis, peptide analysis or electrospray ionization-MS of HOCl-modified apomyoglobin. The latter showed an increase in mass consistent with conversion of the two methionine residues into sulphoxides. A 5-fold excess of HOCl generated approximately three chloramines on the apomyoglobin. These underwent slow decay. Protein carbonyls were formed and were almost entirely located only on the polymer bands. Conversion of positively into negatively charged groups on the protein by succinylation caused preformed aggregates to dissociate. Treatment of apomyoglobin with taurine chloramine generated methionine sulphoxides but few protein carbonyls, and did not result in aggregation. We conclude that aggregation was due to strong, non-covalent interactions between protein chains. We propose that formation of protein carbonyls and possibly chloramines, along with methionine oxidation, alters protein folding to expose hydrophobic areas on neighbouring molecules that associate to form dimers and higher-molecular-mass aggregates. This process could lead to the formation of aggregated proteins at sites of myeloperoxidase activity and contribute to inflammatory tissue injury.

The proteomics of senescence in leaves of white clover, Trifolium repens (L.)

A proteomics approach has been used to study changes in protein abundance during leaf senescence in white clover. Changes in cell ultrastructure were also examined using transmission electron microscopy. The most obvious ultrastructural changes during senescence occurred in chloroplasts, with progressive loss of thylakoid integrity and accumulation of osmiophilic globules in the stroma. Quantitative analysis of 590 leaf protein spots separated by two‐dimensional electrophoresis indicated that approximately 40% of the spots showed significant senescence related changes in abundance. Approximately one‐third of the protein spots present in mature green leaves were also visible by two‐dimensional electrophoresis of an isolated chloroplast fraction, and these spots represented a major proportion of the proteins showing senescence related declines in abundance. Chloroplast proteins that were identified by matrix‐assisted laser desorption/ionization‐time of flight mass fingerprinting included rubisco large and small subunits, a rubisco activase and the 33 kDa protein of the photosystem II oxygen‐evolving complex. These proteins declined in abundance late in senescence, indicating that the photosynthetic apparatus was being degraded. A chloroplast glutamine synthetase showed partial decline in abundance during late senescence but was maintained at levels that may support provision of glutamine for export to other tissues. The results emphasise the importance of proteolysis, chloroplast degradation and remobilisation of nitrogen in leaf senescence.

Evidence for Capsule Switching between Carried and Disease-Causing Neisseria meningitidis Strains

ABSTRACT Changing antigenic structure such as with capsule polysaccharide is a common strategy for bacterial pathogens to evade a host immune system. The recent emergence of an invasive W:2a:P1.7-2,4 sequence type 11 (ST-11) strain of Neisseria meningitidis in New Zealand, an uncommon serogroup/serotype in New Zealand disease cases, was investigated for its genetic origins. Molecular typing of 107 meningococcal isolates with similar serotyping characteristics was undertaken to determine genetic relationships. Results indicated that the W:2a:P1.7-2,4 strain had emerged via capsule switching from a group C strain (C:2a:P1.7-2,4). Neither the upstream nor downstream sites of recombination could be elucidated, but sequence analysis demonstrated that at least 45 kb of DNA was involved in the recombination, including the entire capsule gene cluster. The oatWY gene carried by the W:2a:P1.7-2,4 strain contained the insertion sequence element IS1301, one of five variants of oatWY found in group W135 strains belonging to the carriage-associated ST-22 clonal complex. This suggested that the origin of the capsule genes carried by the invasive W:2a:P1.7-2,4 strain is carriage associated. These results provide novel evidence for the long-standing dogma that disease-associated strains acquire antigenic structure from carriage-associated strains. Moreover, the capsule switch described here has arisen from the exchange of the entire capsule locus.

Profiling the metabolic proteome of bovine mammary tissue

2‐DE and MALDI mass fingerprinting were used to analyse mammary tissue from lactating Friesian cows. The goal was detection of enzymes in metabolic pathways for synthesis of milk molecules including fatty acids and lactose. Of 418 protein spots analysed by PMF, 328 were matched to database sequences, resulting in 215 unique proteins. We detected 11 out of the 15 enzymes in the direct pathways for conversion of glucose to fatty acids, two of the pentose phosphate pathway enzymes and two of the enzymes for lactose synthesis from glucose. We did not detect enzymes that catalyse the first three reactions of glycolysis. Our results are typical of enzyme detection using 2‐DE of mammalian tissues. We therefore advocate caution when relating enzyme abundances measured by 2‐DE to metabolic output as not all relevant proteins are detected. 2‐D DIGE was used to measure interindividual variation in enzyme abundance from eight animals. We extracted relative protein abundances from 2‐D DIGE data and used a logratio transformation that is appropriate for compositional data of the kind represented in many proteomics experiments. Coefficients of variation for abundances of detected enzymes were 3–8%. We recommend use of this transformation for DIGE and other compositional data.

Metabolic proteomics of the liver and mammary gland during lactation

The liver and the mammary gland have complementary metabolic roles during lactation. Glucose synthesized by the liver is released into the circulation and is taken up by the mammary gland where major metabolic products of glucose include milk sugar (lactose) and the glycerol backbone of milk fat (triglycerides). Hepatic synthesis of glucose is often accompanied by β-oxidation in that organ to provide energy for glucose synthesis, while mammary gland synthesizes rather than oxidizes fat during lactation. We have therefore compared enzyme abundances between the liver and mammary gland of lactating Friesian cows where metabolic output is well established. Quantitative differences in protein amount were assessed using two-dimensional differential in-gel electrophoresis. As predicted, the abundances of enzymes catalysing gluconeogenesis and β-oxidation were greatest in the liver, and enzyme abundances in mammary tissue were consistent with fat synthesis rather than β-oxidation.

Fungal epipolythiodioxopiperazine toxins have therapeutic potential and roles in disease

Abstract The epipolythiodioxopiperazine toxins are produced by a wide range of fungi. The compounds in this group inhibit the growth of fungi, RNA viruses and gram-positive bacteria. William Jordan and Stephen Cordiner describe the effects of these on mammalian cells — suppression of normal and tumor cell proliferation, modulation of plasma membrane associated events and inhibition of immune and immune-related functions, for example. There is continuing interest in this group of toxins because of their potential pharmacological value and known or suggested roles in animal and human disease.

Application of proteomics for determining protein markers for wool quality traits.

The technique of two‐dimensional electrophoresis (2‐DE) has been under investigation for its usefulness in identifying protein markers for wool quality traits in sheep. However, before this could be achieved, unique problems relating to the detection and quantitation of wool proteins needed to be overcome so that 2‐DE protein maps could be examined using computational programs like Melanie II. Four protein staining regimes were examined. Colloidal Coomassie Blue G‐250 was found to be superior to Coomassie Blue R‐250 and gave satisfactory staining of all protein classes. Silver staining detects minor strings of keratinous proteins, but unfortunately it negatively stains intermediate filament proteins, the major high sulphur proteins (HSPs) and the high glycine tyrosine proteins and the latter two classes can only be seen by overstaining the background of the gel. In contrast, labeling reduced keratins with [14C]iodoacetamide, followed by autoradiography detection, results in a protein map with low background and all protein spots stained positively. 2‐DE has been used to obtain wool protein maps of Lincoln/Merino chimeric sheep to examine wool originating from two genotypes grown with different crimp frequencies within the same fleece. Between fleece, variations have also been examined. Work to date suggests that several major HSPs may be associated with the fibre curvature trait known as crimp frequency. From matrix‐assisted laser desorption/ionization time‐of‐flight (MALDI‐TOF) mass spectral mapping, one of these proteins has been identified as being from the B2A family from the HSP class.

The Asia Oceania Human Proteome Organisation Membrane Proteomics Initiative. Preparation and characterisation of the carbonate‐washed membrane standard

The Asia Oceania Human Proteome Organisation (AOHUPO) has embarked on a Membrane Proteomics Initiative with goals of systematic comparison of strategies for analysis of membrane proteomes and discovery of membrane proteins. This multilaboratory project is based on the analysis of a subcellular fraction from mouse liver that contains endoplasmic reticulum and other organelles. In this study, we present the strategy used for the preparation and initial characterization of the membrane sample, including validation that the carbonate‐washing step enriches for integral and lipid‐anchored membrane proteins. Analysis of 17 independent data sets from five types of proteomic workflows is in progress.