Validation of nuclear texture, density, morphometry and tissue syntactic structure analysis as prognosticators of cervical carcinoma

OBJECTIVE: To evaluate the performance of karyometry and histometry in the prediction of survival, recurrence and response of early-stage invasive cervical carcinoma. STUDY DESIGN: Nuclear morphometry, chromatin texture and tissue architecture (characterized by syntactic structure analysis) were measured using a semiautomated image analysis system on 46 cases of Feulgen-stained tissue sections. The performance of the features was compared to that of clinical features, reported to be the best prognosticators until now, such as age, lympho-vascular permeation, histologic type, stage and grade. A K nearest neighbor classifier was used for classification. RESULTS: In the prediction of three-year survival, recurrence and response, syntactic structure analysis proved to be the best performer. Classification rates were, respectively, 100%, 94.4% and 94.5%. In all classifications, karyometric and histometric features outperformed clinical features. In general, the best performing features described differences in second-order population statistics (standard deviations). CONCLUSION: The results show that a quantitative analysis based on nuclear morphology, chromatin texture and histology can be considered an excellent aid in the prognosis of invasive cervical carcinoma. The measurements are not hampered by the need to undertake complete resections and are suited to daily practice when implemented in a semiautomated image analysis system.     

Solid state NMR: new tools for insight into enzyme function

NMR has had considerable impact in enzymology, probing evidence for ionization states, conformational 'strain', compressed interactions, electronically unusual species, and conformational dynamics of enzymes. Solid-state NMR is becoming increasingly important in studying enzymes because of a number of recent tools for analysis of proteins by SSNMR, and because of the growing ability to isolate the species of interest for analysis. Here, we review recent studies of a Michaelis complex, of the dynamic functioning of membrane-associated enzymes, and initial studies of several enzymes with redox-active and paramagnetic centers.     

Proteome analysis of human lung squamous carcinoma

Few lung cancer-specific molecular markers have been established in regard of "early-stage" diagnosis and prognosis. In this study the proteome analysis of human lung squamous carcinoma (hLSC) was carried out using two strategies to explore the carcinogenic mechanisms and identify its molecular markers more directly and comprehensively. Comparative proteome analysis on 20 hLSC tissues and paired normal bronchial epithelial tissues revealed 76 differential proteins, among which 68 proteins were identified by PMF. The identified proteins fell into three categories: oncoproteins, cell cycle regulators and signaling molecules. To validate the identified differential proteins, the expressions levels of three differential proteins mdm2, c-jun and EGFR were determined by immunohistochemical staining and immunoblots. The results verified proteome analysis results. Serological proteome analysis (SERPA) of ten hLSC tissues was performed to identify the tumor-associated antigens. The results revealed 36 +/- 8 differential proteins reactive with patients' autologous sera, of which 14 proteins were identified. Six of the 14 proteins, alpha enolase, pre-B cell-enhancing factor precursor, triosephosphate isomerase, phosphoglycerate mutase 1, fructose-bisphosphate aldolase A, and guanine nucleotide-binding protein beta subunit-like protein, were also up-regulated in hLSCs in the comparative proteomic study, which suggests potential application of these 6 hLSC-associated antigens in diagnosis and therapy of hLSC.     

TRPC channels determine human keratinocyte differentiation: new insight into basal cell carcinoma

Aberrant keratinocyte differentiation is considered to be a key mechanism in the onset of hyperproliferative dermatological diseases, including basal cell carcinoma (BCC). It is, therefore, vital to understand what drives keratinocytes to develop such pathological phenotypes. The role of calcium in keratinocyte differentiation is uncontested but the mechanisms controlling calcium-induced differentiation have yet to be completely elucidated. This study was designed to investigate the role of calcium-permeable TRPC channels in human keratinocyte differentiation and BCC, using a combination of molecular and cell biology approaches, involving electrophysiology and Ca(2+)-imaging, on the HaCaT cell line, primary cultures of normal human keratinocytes, and BCC cells. We demonstrated that TRPC1/TRPC4 channel expression was important for keratinocyte differentiation, as knocking out these channels (by siRNA strategy) prevented the induction of Ca(2+)-induced differentiation. TRPC1/TRPC4-mediated calcium entry and endoplasmic reticulum Ca(2+) content increased significantly in differentiated keratinocytes. However, the failure of BCC cells to differentiate was related to a lack of TRPC channel expression and calcium entry. In summary, our data demonstrate that TRPC1 and TRPC4 channels are key elements in keratinocyte Ca(2+) homeostasis and differentiation and may therefore be responsible for skin pathologies.     

Crystal structure of the human N-Myc downstream-regulated gene 2 protein provides insight into its role as a tumor suppressor

Considerable attention has recently been paid to the N-Myc downstream-regulated gene (NDRG) family because of its potential as a tumor suppressor in many human cancers. Primary amino acid sequence information suggests that the NDRG family proteins may belong to the α/β-hydrolase (ABH) superfamily; however, their functional role has not yet been determined. Here, we present the crystal structures of the human and mouse NDRG2 proteins determined at 2.0 and 1.7 Å resolution, respectively. Both NDRG2 proteins show remarkable structural similarity to the ABH superfamily, despite limited sequence similarity. Structural analysis suggests that NDRG2 is a nonenzymatic member of the ABH superfamily, because it lacks the catalytic signature residues and has an occluded substrate-binding site. Several conserved structural features suggest NDRG may be involved in molecular interactions. Mutagenesis data based on the structural analysis support a crucial role for helix α6 in the suppression of TCF/β-catenin signaling in the tumorigenesis of human colorectal cancer, via a molecular interaction.     

Comparative proteomics analysis of human lung squamous carcinoma

Two-dimensional polyacrylamide gel electrophoresis (2-DE) profiles of human lung squamous carcinoma tissue and paired surrounding normal bronchial epithelial tissue were compared. Selected differential protein-spots were identified with peptide mass fingerprinting based on matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) and database searching. Well-resolved and reproducible 2-DE patterns of both the tumor and the normal tissues were acquired. The average deviations of spot position were 0.873+/-0.125mm in IEF direction and 1.025+/-0.213mm in SDS-PAGE direction, respectively. For the tumor tissues, a total of 1349+/-67 spots were detected and 1235+/-48 spots were matched with an average matching rate of 91.5%. For the corresponding normal tissues, a total of 1297+/-73 spots were detected and 1183+/-56 spots were matched with an average matching rate of 91.2%. A total of 1069+/-45 spots were matched between the tumor and the normal tissues. Forty differential proteins between tumor and normal tissues were characterized. Some proteins were the products of oncogenes and others were involved in the regulation of cell cycle and signal transduction. These data are valuable for mass identification of differentially expressed proteins involved in lung carcinogenesis, establishing human lung cancer proteome database and screening molecular marker to further study human lung squamous carcinoma.     

Glutathione content and growth in A549 human lung carcinoma cells

The relationship between glutathione content and cell growth was investigated in A549 human lung carcinoma cells. A decreased cellular glutathione content was achieved by exposing the cells to L-buthionine-SR-sulfoximine (BSO). It also occurred in these cells as they approached their plateau phase of growth. During exponential growth, a lower initial glutathione content correlated with a longer lag phase in subcultured cells. Further, depletion of cellular glutathione by BSO inhibited cell growth. This inhibition became apparent 36 h after the addition of BSO. These observations raise the possibility that a critical concentration of GSH may be required for optimal growth of A549 human lung carcinoma cells.     

Combined physiological and anthropometrical databases as ergonomic tools

The purpose of this study was to establish an ergonomic basis for the integration of female personnel at military workplaces. The results of anthropometrical and physiological measurements of 1337 male and female subjects (aged 18-25 years) are presented. The empirical design included classical and functional body measures of high ergonomic relevance. Additionally, the isometric forearm-flexor and knee-extensor forces of the subjects were tested as representative variables for their physiological suitability. With regard to biometric and strength parameters, the present results clearly show only small overlaps between the sexes. Rating the 5th percentile of the male subjects as an exclusion value, the anthropometrical variables (sitting and standing body height measures, shoulder breadth, hand length and breadth etc.) show that 29% up to 72% of the women do not match this criterion. The situation becomes even worse when strength parameters are taken into consideration: Only 26% (knee extensors) respectively 3% (forearm flexors) of the females generate higher forces than the corresponding 5th percentiles of their male counterparts. The application of the database established in this survey aims at the integration of female personnel at all kinds of military workplaces; our data account for a combination of anthropometrical and physiological variables. In contrast to former systems based on isolated percentile tables, the results of our investigation make multivariate queries possible in optimization processes. This type of combined database is to be considered a useful tool for the evaluation of workplaces and personal equipment, for general ergonomic considerations and for personnel planning purposes to prevent applicants from occupational harm.     

The crystal structure of iron-free human serum transferrin provides insight into inter-lobe communication and receptor binding

Serum transferrin reversibly binds iron in each of two lobes and delivers it to cells by a receptor-mediated, pH-dependent process. The binding and release of iron result in a large conformational change in which two subdomains in each lobe close or open with a rigid twisting motion around a hinge. We report the structure of human serum transferrin (hTF) lacking iron (apo-hTF), which was independently determined by two methods: 1) the crystal structure of recombinant non-glycosylated apo-hTF was solved at 2.7-A resolution using a multiple wavelength anomalous dispersion phasing strategy, by substituting the nine methionines in hTF with selenomethionine and 2) the structure of glycosylated apo-hTF (isolated from serum) was determined to a resolution of 2.7A by molecular replacement using the human apo-N-lobe and the rabbit holo-C1-subdomain as search models. These two crystal structures are essentially identical. They represent the first published model for full-length human transferrin and reveal that, in contrast to family members (human lactoferrin and hen ovotransferrin), both lobes are almost equally open: 59.4 degrees and 49.5 degrees rotations are required to open the N- and C-lobes, respectively (compared with closed pig TF). Availability of this structure is critical to a complete understanding of the metal binding properties of each lobe of hTF; the apo-hTF structure suggests that differences in the hinge regions of the N- and C-lobes may influence the rates of iron binding and release. In addition, we evaluate potential interactions between apo-hTF and the human transferrin receptor.     

New insight into RNase P RNA structure from comparative analysis of the archaeal RNA

A detailed comparative analysis of archaeal RNase P RNA structure and a comparison of the resulting structural information with that of the bacterial RNA reveals that the archaeal RNase P RNAs are strikingly similar to those of Bacteria. The differences between the secondary structure models of archaeal and bacterial RNase P RNA have largely disappeared, and even variation in the sequence and structure of the RNAs are similar in extent and type. The structure of the cruciform (P7-11) has been reevaluated on the basis of a total of 321 bacterial and archaeal sequences, leading to a model for the structure of this region of the RNA that includes an extension to P11 that consistently organizes the cruciform and adjacent highly-conserved sequences.     

Crystal structure of human thimet oligopeptidase provides insight into substrate recognition, regulation, and localization

Thimet oligopeptidase (TOP) is a zinc metallopeptidase that metabolizes a number of bioactive peptides and degrades peptides released by the proteasome, limiting antigenic presentation by MHC class I molecules. We present the crystal structure of human TOP at 2.0-A resolution. The active site is located at the base of a deep channel that runs the length of the elongated molecule, an overall fold first seen in the closely related metallopeptidase neurolysin. Comparison of the two related structures indicates hinge-like flexibility and identifies elements near one end of the channel that adopt different conformations. Relatively few of the sequence differences between TOP and neurolysin map to the proposed substrate-binding site, and four of these variable residues may account for differences in substrate specificity. In addition, a loop segment (residues 599-611) in TOP differs in conformation and degree of order from the corresponding neurolysin loop, suggesting it may also play a role in activity differences. Cysteines thought to mediate covalent oligomerization of rat TOP, which can inactivate the enzyme, are found to be surface-accessible in the human enzyme, and additional cysteines (residues 321,350, and 644) may also mediate multimerization in the human homolog. Disorder in the N terminus of TOP indicates it may be involved in subcellular localization, but a potential nuclear import element is found to be part of a helix and, therefore, unlikely to be involved in transport. A large acidic patch on the surface could potentially mediate a protein-protein interaction, possibly through formation of a covalent linkage.     

Clostridium perfringens: insight into virulence evolution and population structure

Clostridium perfringens is an important pathogen in veterinary and medical fields. Diseases caused by this organism are in many cases life threatening or fatal. At the same time, it is part of the ecological community of the intestinal tract of man and animals. Virulence in this species is not fully understood and it does seem that there is erratic distribution of the toxin/enzyme genes within C. perfringens population. We used the recently developed multiple-locus variable-number tandem repeat analysis (MLVA) scheme to investigate the evolution of virulence and population structure of this species. Analysis of the phylogenetic signal indicates that acquisition of the major toxin genes as well as other plasmid-borne toxin genes is a recent evolutionary event and their maintenance is essentially a function of the selective advantage they confer in certain niches under different conditions. In addition, it indicates the ability of virulent strains to cause disease in different host species. More interestingly, there is evidence that certain normal flora strains are virulent when they gain access to a different host species. Analysis of the population structure indicates that recombination events are the major tool that shapes the population and this panmixia is interrupted by frequent clonal expansion that mostly corresponds to disease processes. The signature of positive selection was detected in alpha toxin gene, suggesting the possibility of adaptive alleles on the other chromosomally encoded determinants. Finally, C. perfringens proved to have a dynamic population and availability of more genome sequences and use of comparative proteomics and animal modeling would provide more insight into the virulence of this organism.     

Crystal structure of group A streptococcus Mac-1: insight into dimer-mediated specificity for recognition of human IgG

Group A Streptococcus secretes cysteine proteases named Mac-1 and Mac-2 that mediate host immune evasion by targeting both IgG and Fc receptors. Here, we report the crystal structures of Mac-1 and its catalytically inactive C94A mutant in two different crystal forms. Despite the lack of sequence homology, Mac-1 adopts the canonical papain fold. Alanine mutations at the active site confirmed the critical residues involved in a papain-like catalytic mechanism. Mac-1 forms a symmetric dimer in both crystal forms and displays the unique dimer interface among papain superfamily members. Mutations at the dimer interface resulted in a significant reduction in IgG binding and catalysis, suggesting that the dimer contributes to both IgG specificity and enzyme cooperativity. A tunnel observed at the dimer interface constitutes a target for designing potential Mac-1-specific antimicrobial agents. The structures also offer insight into the functional difference between Mac-1 and Mac-2.     

Spliceosomal introns as tools for genomic and evolutionary analysis

Over the past 5 years, the availability of dozens of whole genomic sequences from a wide variety of eukaryotic lineages has revealed a very large amount of information about the dynamics of intron loss and gain through eukaryotic history, as well as the evolution of intron sequences. Implicit in these advances is a great deal of information about the structure and evolution of surrounding sequences. Here, we review the wealth of ways in which structures of spliceosomal introns as well as their conservation and change through evolution may be harnessed for evolutionary and genomic analysis. First, we discuss uses of intron length distributions and positions in sequence assembly and annotation, and for improving alignment of homologous regions. Second, we review uses of introns in evolutionary studies, including the utility of introns as indicators of rates of sequence evolution, for inferences about molecular evolution, as signatures of orthology and paralogy, and for estimating rates of nucleotide substitution. We conclude with a discussion of phylogenetic methods utilizing intron sequences and positions.     

Multiple sequence alignments as tools for protein structure and function prediction

Multiple sequence alignments have much to offer to the understanding of protein structure, evolution and function. We are developing approaches to use this information in predicting protein-binding specificity, intra-protein and protein-protein interactions, and in reconstructing protein interaction networks.     

New insight into the structure and function of the alternative oxidase

The alternative oxidase is a ubiquinol oxidase found in plant mitochondria, as well as in the mitochondria of some fungi and protists. It catalyzes a cyanide-resistant reduction of oxygen to water without translocation of protons across the inner mitochondrial membrane, and thus functions as a non-energy-conserving member of the respiratory electron transfer chain. The active site of the alternative oxidase has been modelled as a diiron center within a four-helix bundle by Siedow et al. (FEBS Lett. 362 (1995) 10-14) and more recently by Andersson and Nordlund (FEBS Lett. 449 (1999) 17-22). The cloning of the Arabidopsis thaliana IMMUTANS (Im) gene, which encodes a plastid enzyme distantly related to the mitochondrial alternative oxidases (Wu et al. Plant Cell 11 (1999) 43-55; Carol et al. Plant Cell 11 (1999) 57-68), has now narrowed the range of possible ligands to the diiron center of the alternative oxidase. The Im protein sequence suggests a minor modification to the recent model of the active site of the alternative oxidase. This change moves an invariant tyrosine into a conserved hydrophobic pocket in the vicinity of the active site, in a position analogous to the long-lived tyrosine radical at the diiron center of ribonucleotide reductase, and similar to the tyrosines near the diiron center of bacterioferritin and rubrerythrin. The Im sequence and modified structural model yield a compelling picture of the alternative oxidase as a diiron carboxylate protein. The current status of the relationship of structure to function in the alternative oxidase is reviewed.     

Ab initio structure of human seminal plasma prostatic inhibin gives significant insight into its biological functions

Human seminal plasma prostatic inhibin (HSPI) is a protein isolated from the human prostate gland. Despite its profound biomedical and biotechnological importance, the 3D structure of this protein of 94 amino acids remains undeciphered. The difficulties in extracting it in pure form and crystallizing it have restrained the determination of its structure experimentally. The homology-based computational methods are also not applicable, as HSPI lacks sufficient sequence homology with known structures in the protein data banks. We have predicted the structure of HSPI by a knowledge-based method using nonparametric multivariate statistical techniques. Stereochemical and other standard validation tests confirm this to be a well-refined structure. The biophysical properties exhibited by this structure are in good agreement with the NMR experimental observations. Docking and other computational studies on this structure provide significant explanation and insight into its binding activities and related biological and immunogenic functions and offer new directions for its potential applications.     

Combined experimental and numerical analysis of the flow structure into the left ventricle

Fluid dynamics is used for diagnosis in cardiology only to a partial extent. Indeed several aspects of cardiac flows and their relation with pathophysiology are unknown. The flow that develops into the left ventricle is here studied by using a combination of numerical and experimental models. The former allows a detailed three-dimensional analysis, the latter can be used in conditions, like in presence of turbulence, that are out of reach of the current computational power. The three-dimensional flow dynamics is analyzed in terms of its vortical structure. The study, within its limitations, provides further physical understanding about the intraventricular flow structure. This could eventually support the development of cardiac diagnostic indicators based on fluid dynamics.     

Arginase and ornithine, as markers in human non-small cell lung carcinoma

The arginase activity and ornithine level were determined in tissue obtained from patients with non-small cell lung carcinoma (NSLC). The arginase activity and ornithine level in tumor tissues were 1.89 +/- 1.28 U/mg protein and 42.32 +/- 25.82 nmol/mg protein, respectively versus 0.67 +/- 0.19 U/mg protein and 10.12 +/- 3.69 nmol/mg protein for normal tissues (p < 0.01).