Detection of renal cell carcinoma-associated markers via proteome- and other 'ome'-based analyses.

Proteome analysis has rapidly developed in the post-genome era and is now widely accepted as the complementary technology for genetic profiling. It has been shown to be a powerful tool for studying human diseases and for identifying novel prognostic, diagnostic and therapeutic markers. This review focuses on the identification of new biomarkers and therapeutic targets for renal cell carcinoma using different 'ome'-based technologies.     

Birth Weight and Its Relationship to Size Attained and Relative Fat Distribution at 7 to 12 Years of Age

The relationship between birth weight and relative subcutaneous fat distribution at school age was considered in 131 boys and 106 girls 7 to 12 years of age. Relative fat distribution at school age was estimated with the ratio of the subscapular to triceps skinfolds (S/T) for the total sample, and with the ratio of the sum of two trunk (subscapular, midaxillary) to the sum of two extremity (triceps, medial calf) skinfolds (T/E) for subsamples of 102 boys and 63 girls. There were no sex differences in the S/T ratio (mm/mm), boys 0.62 ± 0.15, girls 0.63 ± 0.18; T/E ratio (mm/mm), boys 0.58 ± 0.13, girls 0.59 ± 0.16; and BMI (kg/m²), boys 17.1 ± 2.4, girls 16.9 ± 2.2. Second order partial correlations, controlling for age and the BMI or age and sum of skinfolds, between birth weight and the skinfold ratios are, respectively, ?0.22 and ?0.20 (p<0.01) for S/T and ?0.29 and ?032 (p<0.01) for T/E in girls, and ?0.18 and ?0.17 (p<0.05) for S/T and ?0.06 and ?0.6 for T/E in boys. Though low, the correlations suggest that as birth weight decreases proportionally more subcutaneous fat is accumulated on the trunk than on the extremities, more so in females than in males. Results of stepwise multiple regression analyses indicate that birth weight accounts for from 2% to 8% of the variance in relative subcutaneous fat distribution at school age.     

Actinomycete diversity associated with foaming in activated sludge plants

Large numbers of mycolic acid-containing actinomycetes were isolated from foam and scum samples taken from three activated-sludge sewage-treatment plants using several selective isolation media. Organisms presumptively identified as gordonae formed the dominant population in all of the samples. A representative set of these strains have chemical properties consistent with their classification in the genusGordona. Forty-eight of theGordona strains were compared through 165 unit characters with the type strains of validly described species ofGordona. The resultant data were examined using the Jaccard and simple matching coefficients and clustering achieved using the unweighted pair group method with arithmetic averages algorithm. The numerical classification was only marginally affected by the statistics used or by test error, estimated as 3.92%. The isolates were assigned to five multi-membered and 28 single-membered clusters defined by the simple matching coefficient at the 89% similarity level. With few exceptions, the isolates were sharply separated from theGordona marker strains. Essentially the same classification was obtained when the test strains were examined using a Curie-point pyrolysis mass spectrometric procedure. It can be concluded that the gordonae form a heterogeneous taxonomic group, the members of which can be distinguished from representatives of validly described species ofGordona.     

Submitochondrial particles asin vitro biosensors of heavy metal toxicity

The effects on mitochondrial respiratory parameters of heavy metals, such as Cu, Ni, Pb, Cd, Zn, Ag, Hg, were recorded by using thein vitro response of submitochondrial particles (SMP) from beef heart mitochondria.The toxicity of these elements was estimated by determining their effects on the energy-coupled reverse electron transfer (RET), which is induced by ATP and succinate at first site level of the respiratory chain in SMP.The RET rate was easily monitored by recording spectrophotometrically at 340 nm the production of NADH, arising from the reduction of exogenous NAD⁺ by RET.The toxicity values were expressed as the toxicant molar concentration which decreases the rate of reduction of NAD⁺ to an extent of 50 percent (EC50). The toxicity increased in the following order: Ni²⁺2+2+< Cd²⁺2+2++.The SMP data were compared with the toxicity values obtained from a variety of biological systems currently used for toxicity testing. The results obtained demonstrate that the SMP test generally provides a good estimate of metal toxicity for several fish and invertebrate species. This is demonstrated by the statistical parameters obtained in the regression analysis. The broadened 95% confidence intervals and, in particular, the poor correlations obtained for some aquatic organisms can be ascribed to the more complex metabolic interactions and competing toxic pathways in aquatic organisms, when compared to SMP.     

含吸氢酶基因（hup）嵌合质粒的构建和吸氢酶活性在阴沟肠杆菌中的表达

以质粒ｐＲＫ４０４为载体亚克隆含大豆根瘤菌吸氢酶结构基因（ｈｕｐ）的片段,构建成嵌合质粒ｐＨＲ１１、ｐＨＲ４和ｐＨＲ１０。通过三亲本杂交将这些嵌合质粒导入无吸氢活性的Ｒｈｏｄｏｂａｃｔｅｒｓｐｈａｅｒｏｉｄｅｓ２４１菌株（Ｈｕｐ－）,均获得Ｈｕｐ＋的接合子。利用启动子检测质粒ｐＭＰ２２０证明,在ｈｕｐ对结构基因上游１．２ｋｂ内存在ｈｕｐ启动基因片段。以ｐＲＫ２０１３为助质粒可将ｐＨＲ１１导入Ｅｎｔｅｒｏｂａｃｔｅｒｃｌｏａｃａｅ和Ｋｌｅｂｓｉｅｌｌａｏｘｙｔｏｃａ等土壤固氮菌株。本文以接合子Ｅ．ｃｌｏａｃａｅＥＨ１１１３为例,通过对基因组ＤＮＡＳｏｕｔｈｅｒｎ杂交分析证明,嵌合质粒ｐＨＲ１１在ＥＨ１１１３中稳定贮存和复制。Ｈ２诱导接合子ＥＨ１１１３吸氢酶活性高表达,吸氢活性与放氢活性比值约为对照的两倍。当以延胡索酸为电子受体时,吸氢酶的吸氢作用支持菌株固氮酶活性的提高。     

A Major Gene for Resting Metabolic Rate Unassociated with Body Composition: Results from the Québec Family Study

A major gene hypothesis for resting metabolic rate (RMR) was investigated using segregation analysis (POINTER) of data on families participating in Phase 2 of the Québec Family Study. Complete analyses were conducted on RMR adjusted for age, and also on RMR adjusted for age and other covariates, primarily fat mass (FM) and fat-free mass (FFM). Prior to adjustment for covariates, support for a major gene hypothesis was equivocal — i.e., there was evidence for either a major gene or a multifactorial component (i.e., polygenic and/or familial environment). The multifactorial model was preferred over the major gene model, although the latter did segregate according to Mendelian expectations. However, after the effects of FM and FFM were accounted for, a major gene effect was unambiguous and compelling. The putative locus accounted for 57% of the variance, affected 7% of the sample, and led to high values of RMR. The lack of a significant multifactorial effect suggested that the familial etiology of RMR adjusted for FM and FFM was likely to be entirely a function of the major locus. Comparing the RMR results from pre- and post-adjustment for FM and FFM suggests a plausible hypothesis. We know from earlier studies in this sample that there is a putative major gene for FM and a major non-Mendelian effect for FFM. The current study leads us to speculate that: (1) the gene(s) affecting body size and body composition also may have an effect on RMR, and further (2) removal of the effect of the major gene(s) for body size and composition allowed for detection of an additional major gene affecting only the RMR. Thus, RMR appears to be an oligogenic trait.     

Significant hydrogen exchange protection in GroEL-bound DHFR is maintained during iterative rounds of substrate cycling.

An unresolved key issue in the mechanism of protein folding assisted by the molecular chaperone GroEL is the nature of the substrate protein bound to the chaperonin at different stages of its reaction cycle. Here we describe the conformational properties of human dihydrofolate reductase (DHFR) bound to GroEL at different stages of its ATP-driven folding reaction, determined by hydrogen exchange labeling and electrospray ionization mass spectrometry. Considerable protection involving about 20 hydrogens is observed in DHFR bound to GroEL in the absence of ATP. Analysis of the line width of peaks in the mass spectra, together with fluorescence quenching and ANS binding studies, suggest that the bound DHFR is partially folded, but contains stable structure in a small region of the polypeptide chain. DHFR rebound to GroEL 3 min after initiating its folding by the addition of MgATP was also examined by hydrogen exchange, fluorescence quenching, and ANS binding. The results indicate that the extent of protection of the substrate protein rebound to GroEL is indistinguishable from that of the initial bound state. Despite this, small differences in the quenching coefficient and ANS binding properties are observed in the rebound state. On the basis of these results, we suggest that GroEL-assisted folding of DHFR occurs by minor structural adjustments to the partially folded substrate protein during iterative cycling, rather than by complete unfolding of this protein substrate on the chaperonin surface.     

Possible role of a short extra loop of the long-chain flavodoxin from Azotobacter chroococcum in electron transfer to nitrogenase: Complete 1H, 15N and 13C backbone assignments and secondary solution structure of the flavodoxin

Summary The ¹H, ¹⁵N and ¹³C backbone and ¹H and ¹³C beta resonance assignments of the long-chain flavodoxin from Azotobacter chroococcum (the 20-kDa nifF product, flavodoxin-2) in its oxidized form were made at pH 6.5 and 30°C using heteronuclear multidimensional NMR spectroscopy. Analysis of the NOE connectivities, together with amide exchange rates, ³J_Hn_H coupling constants and secondary chemical shifts, provided extensive solution secondary structure information. The secondary structure consists of a five-stranded parallel -sheet and five -helices. One of the outer regions of the -sheet shows no regular extended conformation, whereas the outer strand 4/6 is interrupted by a loop, which is typically observed in long-chain flavodoxins. Two of the five -helices are nonregular at the N-terminus of the helix. Loop regions close to the FMN are identified. Negatively charged amino acid residues are found to be mainly clustered around the FMN, whereas a cluster of positively charged residues is located in one of the -helices. Titration of the flavodoxin with the Fe protein of the A. chroococcum nitrogenase enzyme complex revealed that residues Asn¹¹, Ser⁶⁸ and Asn⁷² are involved in complex formation between the flavodoxin and Fe protein. The interaction between the flavodoxin and the Fe protein is influenced by MgADP and is of electrostatic nature.Abbreviations SQ semiquinone - FMN riboflavin 5-monophosphate; nif, nitrogen fixation - TSP 3-(trimethylsilyl)propionate sodium salt - DSS 2,2-dimethyl-2-silapentane-5-sulfonate sodium salt Supplementary Material is available on request, comprising a Materials and Methods section for the expression and purification of the A. chroococcum flavodoxin, a Table S1 containing the parameters of the titration of A. chroococcum flavodoxin with the Fe protein, and a Table S2 containing the ¹⁵N, H^N, ¹³C, ¹H, ¹³C, ¹H and ¹³CO chemical shifts.To whom correspondence should be addressed.     

Kinetic studies of electron transfer in the cyt b 5 : metHb system

 The kinetics of methemoglobin reduction by cytochrome b ₅ has been studied by stopped-flow and saturation transfer NMR. A forward rate constant k _f = 2.44×10⁴ M^–1 s^–1 and a reverse rate constant k _b = 540 M^–1s^–1 have been observed at 10 mm, pH 6.20, 25  °C. The ratio k _f/k _b = k _eq = 43.6 is in good agreement with the equilibrium constant calculated from the electrochemical potential between cyt b ₅ and methemoglobin. A bimolecular collisional mechanism is proposed for the electron transfer from cyt b ₅ to methemoglobin based on the kinetic data analysis. The dependence of the rate constants on ionic strengths supports such collisional mechanism. It is also found that the reaction rate strongly depends on the conformations of methemoglobin. Received: 20 February 1996 / Accepted: 4 June 1996