Natural Larval Diet Differently Influences the Pattern of Developmental Changes in DNA 5-Methylcytosine Levels in Apis mellifera Queens as Compared with Workers and Drones.

The principal mechanism of gene activation/silencing is DNA 5-methylcytosine methylation. This study was aimed at determining global DNA methylation levels in larvae, prepupae, pupae, and 1-day-old adults of Apis mellifera queens, workers and drones. The Imprint Methylated DNA Quantification Kit MDQ1 was used. Percentages of DNA 5-methylcytosine were low and relatively similar in the larvae of all the castes until 4th day of larval development (3-5%). However, they were higher in the drone and worker larvae than in the queen larvae. Generally, the developmental patterns of changes in the DNA methylation levels were different in the queens in comparison with the drones and workers. While methylation increased in the queens, it decreased in the drones and workers. Methylated DNA methylcytosine percentages and weights in the queen prepupae (15%, 9.18 ng) and pupae (21%, 10.74 ng) were, respectively, three and four times higher than in the worker/drone brood of the same age (2.5-4%, 0.03-0.07 ng). Only in the queens, after a substantial increase, did DNA methylation decrease almost twice between the pupal stage and queen emergence (from 21% and 10.74 ng to 12% and 6.78 ng). This finding seems very interesting, particularly for experimental gerontology.

Unexpectedly strong effect of caffeine on the vitality of western honeybees (Apis mellifera).

We examined the influence of caffeine on honeybee lifespan, Nosema resistance, key enzyme activities, metabolic compound concentrations, and total DNA methylation levels. Caffeine slowed age-related metabolic tendencies. Bees that consumed caffeine lived longer and were not infested with Nosema spp. Caffeine-treated workers had higher protein concentrations. The levels increased with aging but they then decreased in older bees. Caffeine increased the activities of antioxidant enzymes (SOD, GPx, CAT, GST), AST, ALT, ALP, neutral proteases, and protease inhibitors, and the concentrations of uric acid, triglycerides, cholesterol, glucose, and Ca2+. Acidic and alkaline protease activities were lower in the bees treated with caffeine. Creatinine and Mg2+ concentrations were higher in the caffeine-treated workers but only up to 14 days of age. Caffeine significantly decreased DNA methylation levels in older bees. The compound could be considered as a natural diet supplement increasing apian resistance to stress factors. Our studies will enhance possibilities of using Apis mellifera as a model organism in gerontological studies.

A novel human gene (SARM) at chromosome 17q11 encodes a protein with a SAM motif and structural similarity to Armadillo/beta-catenin that is conserved in mouse, Drosophila, and Caenorhabditis elegans.

A novel human gene, SARM, encodes the orthologue of a Drosophila protein (CG7915) and contains a unique combination of the sterile alpha (SAM) and the HEAT/Armadillo motifs. The SARM gene was identified on chromosome 17q11, between markers D17S783 and D17S841 on BAC clone AC002094, which also included a HERV repeat and keratin-18-like, MAC30, TNFAIP1, HSPC017, and vitronectin genes in addition to three unknown genes. The mouse SARM gene was located on a mouse chromosome 11 BAC clone (AC002324). The SARM gene is 1.8 kb centromeric to the vitronectin gene, and the two genes share a promoter region that directs a high level of liver-specific expression of both the SARM and the vitronectin genes. In addition to the liver, the SARM gene was highly expressed in the kidney. A 0.4-kb antisense transcript was coordinately expressed with the SARM gene in the kidney and liver, while in the brain and malignant cell lines, it appeared independent of SARM gene transcription. The SARM gene encodes a protein of 690 amino acids. Based on amino acid sequence homology, we have identified a SAM motif within this derived protein. Structure modeling and protein folding recognition studies confirmed the presence of alpha-alpha right-handed superhelix-like folds consistent with the structure of the Armadillo and HEAT repeats of the beta-catenin and importin protein families. Both motifs are known to be involved in protein-protein interactions promoting the formation of diverse protein complexes. We have identified the same conserved SAM/Armadillo motif combination in the mouse, Drosophila, and Caenorhabditis elegans SARM proteins.

[Morphometric features of tarsus in Dermacentor reticulatus (Fabricius, 1794) (Acari: Ixodida: Ixodidae) larvae from Polish and Slovakian populations]. / Cechy morfometryczne stóp larw Dermacentor reticulatus (Fabricius, 1794) (Acari: Ixodida: Ixodidae) z populacji Polskiej i Slowackiej.

Dermacentor reticulatus is widely distributed dangerous tick that usually lives in the river valleys, boggy forests, meadows, and wooded pastures. Tick populations from various regions may exhibit morphological differences. In our study we compared morphometric features of tarsus in larvae D. reticulatus from Polish and Slovakian populations. I tarsus width, III tarsus length, and length of dorsal setae of I tarsus were significantly higher in Polish populations. Indices of width to length of tarsus I and tarsus III were also significantly different in both populations. The other examined morphologic features were similar, what may result from the same environmental conditions of both populations.

How universal are fold recognition parameters. A comprehensive study of alignment and scoring function parameters influence on recognition of distant folds.

A choice of sequence-structure similarity scoring function parameters can significantly alter results of the performance of the recognition of distantly related folds. It therefore constitutes a critical part of fold recognition process. In order to increase an understanding of the influence of parameter choice, a comprehensive benchmark of very hard (SFOLD) and medium hard (SFAM) fold recognition examples has been derived from the SCOP database of protein structure families. These benchmarks have subsequently been used to optimize, validate and analyze dependence of recognition sensitivity on alignment and fold similarity score parameters for different scoring functions. Significant variation of the common parameters has been observed for different functions, leading to the conclusion that optimal parameter sets are not universal. The scope of solutions common to any pair of scoring function is relatively small, hence, using jury method for fold prediction seems not appropriate. Also, using a redundant version of fold libraries significantly increases odds of identification of distantly related fold.

From fold recognition to homology modeling: an analysis of protein modeling challenges at different levels of prediction complexity.

An analysis of different approaches to protein structure prediction is presented based solely on the range of models submitted to the third Critical Assessment of Protein Structure Prediction (CASP3) conference. CASP conferences evaluate the current state of the art of protein structure prediction by comparing blind prediction efforts of many groups for the same set of target sequences. Target sequences may be highly similar to those with known structure or can be totally (at least superficially) sequentially dissimilar. Techniques applied to those blind predictions (over 40 targets) ranges from a detailed homology prediction to the detection of remote homologues well below a twilight zone of protein sequence similarity. For the CASP3 conference, we have submitted predictions, totaling 35, with various levels of difficulty and complexity. For ten submitted homology targets, eight of them were determined by experiment so far. The RMSD of C-alpha atoms are 1.2-1.7, 2.3, and 4.6-17.9 A for the three easy targets, two hard targets, and three very hard homology targets, respectively. Out of 18-fold recognition predictions available for analysis, we got six correct predictions, five near misses, three tough near misses and four far misses. Here we analyze successes and failures of those predictions in an attempt to identify common problems and common achievements.

NMR relaxation time studies of large bowel neoplasms.

The purpose of the study was to determine if measurements of the NMR relaxation times could be useful in distinguishing healthy from neoplasmatic tissue in in vitro study of the slices from the large bowel obtained during surgery before histological examination. Tissue samples taken from the center of the tumor and from the distal part of the excised large bowel segment were stored at 4 degrees C in closed tubes not longer than 24 hours. The measurements were performed at 37 degrees C using a pulse spectrometer operating at 27 MHz. After NMR investigation all the tissue samples were preserved and carefully examined by conventional histopathological techniques. Both for the T1 and the T2 the mean values of the relaxation time for the respective neoplasmatically changed group and normal, non-changed group, were statistically different. However, the differences in measured relaxation times are too small to use the NMR method alone for diagnosis. The NMR method can be useful only for an initial selection of tissue samples with possible cancer changes but histopathological examination is required to verify that cancer is present in the tissue.

Folding simulations and computer redesign of protein A three-helix bundle motifs.

In solution, the B domain of protein A from Staphylococcus aureus (B domain) possesses a three-helix bundle structure. This simple motif has been previously reproduced by Kolinski and Skolnick (Proteins 18: 353-366, 1994) using a reduced representation lattice model of proteins with a statistical interaction scheme. In this paper, an improved version of the potential has been used, and the robustness of this result has been tested by folding from the random state a set of three-helix bundle proteins that are highly homologous to the B domain of protein A. Furthermore, an attempt to redesign the B domain native structure to its topological mirror image fold has been made by multiple mutations of the hydrophobic core and the turn region between helices I and II. A sieve method for scanning a large set of mutations to search for this desired property has been proposed. It has been shown that mutations of native B domain hydrophobic core do not introduce significant changes in the protein motif. Mutations in the turn region were also very conservative; nevertheless, a few mutants acquired the desired topological mirror image motif. A set of all atom models of the most probable mutant was reconstructed from the reduced models and refined using a molecular dynamics algorithm in the presence of water. The packing of all atom structures obtained corroborates the lattice model results. We conclude that the change in the handedness of the turn induced by the mutations, augmented by the repacking of hydrophobic core and the additional burial of the second helix N-cap side chain, are responsible for the predicted preferential adoption of the mirror image structure.

The hydration of proteins in solutions by self-diffusion coefficients. NMR study.

The hydration of the globular (lysozyme, albumin) and fibrillar (fibrinogen) proteins in solution has been determined from the measurements of the self-diffusion coefficient by NMR pulsed gradient method. It has been concluded that the concentration dependencies of the self-diffusion coefficient of water molecules in protein solution are controlled by two additive factors: obstruction effect correlated with the protein dimension and direct hydration effect proportional to the number of the adsorption sites on the protein surface.

Does a backwardly read protein sequence have a unique native state?

Amino acid sequences of native proteins are generally not palindromic. Nevertheless, the protein molecule obtained as a result of reading the sequence backwards, i.e. a retro-protein, obviously has the same amino acid composition and the same hydrophobicity profile as the native sequence. The important questions which arise in the context of retro-proteins are: does a retro-protein fold to a well defined native-like structure as natural proteins do and, if the answer is positive, does a retro-protein fold to a structure similar to the native conformation of the original protein? In this work, the fold of retro-protein A, originated from the retro-sequence of the B domain of Staphylococcal protein A, was studied. As a result of lattice model simulations, it is conjectured that the retro-protein A also forms a three-helix bundle structure in solution. It is also predicted that the topology of the retro-protein A three-helix bundle is that of the native protein A, rather than that corresponding to the mirror image of native protein A. Secondary structure elements in the retro-protein do not exactly match their counterparts in the original protein structure; however, the amino acid side chain contract pattern of the hydrophobic core is partly conserved.

Nuclear relaxation in serum protein mixtures and serum samples.

The Nuclear Magnetic Relaxation Dispersion (NMRD) profiles of water protons in aqueous solutions containing various total amounts of human serum protein (5-14 pcw) in identical macro molecular ratios (60% of albumin, 20% of gamma globulins, and 20% of other globulins) were studied to examine the influence of the individual relaxation properties of the proteins on the global relaxation time of their mixture. The "synthetic" profiles calculated from the single protein relaxivities overlap with the experimental NMRD data for protein mixtures; similar results were obtained for the relaxation rates measured at 27 MHz of the serum samples from patients with different cancer diseases. The results confirm that the global relaxation of serum is determined by the relaxation of the basic serum proteins.

Plasminogen activators and their inhibitors in normal, hyperplastic and carcinomatous human endometrium.

Plasminogen activators (PA) and inhibitors of urokinase were determined by a solid phase 125I-fibrin assay in endometrial tissue homogenates from 87 patients. PA was also determined by a histochemical method. Patients were divided according to histopathological diagnosis into three groups; normal, hyperplastic and cancerous. The mean values and S.D. of PA in control endometria, in hyperplastic endometria and in endometrial cancer were 0.68 +/- 0.55 units per mg protein, 1.9 +/- 1.6 units per mg protein and 3.21 +/- 1.03 units per mg protein, respectively. The results of the histochemical assay of PA correlated with the results of 125I-fibrin assay (R = 0.818, p less than or equal to 0.001). The relative PA activity of urokinase-type was the lowest in normal endometrium; it increased in hyperplastic and it was the highest in carcinomatous endometrium. The urokinase inhibitor activity was similar in control and carcinomatous groups; it was slightly but significantly higher in hyperplasia. The results support the contention that PA reflects malignant transformation of endometrial cells. We suggest that determination of PA may facilitate diagnosis and proper treatment of precancerous and cancerous states of endometrium.

A study of the freezing of water in human uterine muscle by proton magnetic resonance.

Human uterine muscle and its nuclear fractions have been studied by means of nuclear magnetic resonance at temperatures from 300 degrees K to 143 degrees K. Different proton populations have been detected above and below the freezing point. On this basis it is suggested that the freezing of water in uterine muscle starts at the cell nuclei.