The role of AtMUS81 in DNA repair and its genetic interaction with the helicase AtRecQ4A.

The endonuclease MUS81 has been shown in a variety of organisms to be involved in DNA repair in mitotic and meiotic cells. Homologues of the MUS81 gene exist in the genomes of all eukaryotes, pointing to a conserved role of the protein. However, the biological role of MUS81 varies between different eukaryotes. For example, while loss of the gene results in strongly impaired fertility in Saccharomyces cerevisiae and nearly complete sterility in Schizosaccharomyces pombe, it is not essential for meiosis in mammals. We identified a functional homologue (AtMUS81/At4g30870) in the genome of Arabidopsis thaliana and isolated a full-length cDNA of this gene. Analysing two independent T-DNA insertion lines of AtMUS81, we found that they are sensitive to the mutagens MMS and MMC. Both mutants have a deficiency in homologous recombination in somatic cells but only after induction by genotoxic stress. In contrast to yeast, no meiotic defect of AtMUS81 mutants was detectable and the mutants are viable. Crosses with a hyperrecombinogenic mutant of the AtRecQ4A helicase resulted in synthetic lethality in the double mutant. Thus, the nuclease AtMUS81 and the helicase AtRecQ4A seem to be involved in two alternative pathways of resolution of replicative DNA structures in somatic cells.

Identification of post-translationally modified proteins in proteome studies.

Proteome studies are powerful tools to solve many different problems in metabolism, signal transduction, drug discovery, and other areas of interest in life sciences. Up to now, high-sensitive methods for protein identification after two-dimensional gel electrophoresis using mass spectrometry are available. However, the identification of post-translational modifications after two-dimensional gel electrophoresis is still an unsolved problem. In this paper, we want to give several examples for the successful identification of post-translational modifications and point mutations.

The ATP-binding site of brain phosphatidylinositol 4-kinase PI4K230 as revealed by 5'-p-fluorosulfonylbenzoyladenosine.

The ATP-binding site of purified bovine brain phosphatidylinositol 4-kinase 230 (PI4K230) was studied by its reaction with 5'-p-fluorosulfonylbenzoyladenosine (FSBA), an ATP-like alkylating reagent. Four hundred to eight hundred micromolar FSBA inactivated PI4K230 specifically with apparently first-order kinetics and resulted in 50% loss of enzyme activity in 36--130 min. The specificity of the reaction with FSBA was demonstrated by the lack of inactivation with 5'-p-fluorosulfonylbenzoyl chloride and by protection with ATP and ATP analogues against inactivation. Most ATP analogues competed with FSBA inactivation in order of their increasing hydrophobicity, parallel to their inhibitory potency in activity measurements. The specific binding of FSBA to PI4K230 was demonstrated also by Western-blot experiments. These results suggest that FSBA-reactive group(s) involved in the enzyme activity are located near to the ATP-binding site in a hydrophobic region of native PI4K230. Experiments with site-directed mutagenesis indicate that the conserved Lys-1792 plays essential role in the enzyme activity and serves as one target of affinity labelling by FSBA. Prevention of both Lys-1792-directed and Lys-1792-independent binding of FSBA by Cibacron Blue 3GA suggest that these sites are located spatially close to each other.

Human phosphatidylinositol 4-kinase isoform PI4K92. Expression of the recombinant enzyme and determination of multiple phosphorylation sites.

Human phosphatidylinositol 4-kinase, isoform PI4K92, was expressed as His6 tagged protein in Sf9 cells reaching a level of approximately 5% of cellular protein. The enzyme can be purified nearly to homogeneity in a single step by absorption/desorption on Ni/nitriloacetic acid agarose magnetic beads. High Km values in the millimolar range for ATP and PtdIns as well as only a moderate inhibition by adenosine and a sensitivity to Wortmannin (IC50 approximately 300 nM) characterize the enzyme as a type 3 PI4K. The enzyme produces PtdIns4P as product. The isolated enzyme is a phosphoprotein, additionally phosphate is incorporated by incubation with ATP/Mg or ATP/Mn. Phosphorylation sites were mapped by MALDI-MS and LC-MS/MS at the following positions: S258, T263, S266, S277, S294, T423, S496, T504. Accordingly, a stretch of 81 amino acids between the common and the C-terminal catalytic domain was designated phosphorylation domain.

Tissue and serum sialidase levels in breast cancer.

Breast cancer is both one of the most common and one of the most treatable of all human malignancies. It has been suggested by various investigators that sialic acid increases in the sera of cancerous patients. In cancer patients, an increase in the levels of serum sialic acid may also be due to an increase in the activity of serum or tissue sialidase. The purpose of the present investigation was to determine whether the concentration of sialidase in serum and breast tissue could be used as a tumor marker in breast cancer. In this study; serum sialidase levels in 26 patient with breast cancer and 31 controls were found to be 77.04+/-25.07 U/l and 55.56+/-7.50 U/l, respectively. The mean tissue sialidase levels in 26 breast cancer patients and 13 controls were 39.76+/-17.03 U/g protein and 14.30+/-7.09 U/g protein, respectively. Serum and tissue sialidase levels in breast cancer were significantly higher than those found in the control group (P < 0.001). The mean serum and tissue sialidase levels in 14 Grade I-II and 12 Grade III breast cancer patients were found to be 67.73+/-11.87 U/l and 33.41+/-12.17 U/g protein and 87.89+/-31.94 U/l and 47.17+/-19.30 U/g protein, respectively. Also we found a significant difference between the levels of serum and tissue sialidase in Grade I-II and III (P < 0.05).

Functional expression and characterisation of a new human phosphatidylinositol 4-kinase PI4K230.

By constructing DNA probes we have identified and cloned a human PtdIns 4-kinase, PI4K230, corresponding to a mRNA of 7.0 kb. The cDNA encodes a protein of 2044 amino acids. The C-terminal part of ca. 260 amino acids represents the catalytic domain which is highly conserved in all recently cloned PtdIns 4-kinases. N-terminal motifs indicate multiple heterologous protein interactions. Human PtdIns 4-kinase PI4K230 expressed in vitro exhibits a specific activity of 58 micromol mg-1min-1. The enzyme expressed in Sf9 cells is essentially not inhibited by adenosine, it shows a high Km for ATP of about 300 microM and it is half-maximally inactivated by approximately 200 nM wortmannin. These data classify this enzyme as type 3 PtdIns 4-kinase. Antibodies raised against the N-terminal part moderately activate and those raised against the C-terminal catalytic domain inhibit the enzymatic activity. The coexistence of two different type 3 PtdIns 4-kinases, PI4K92 and PI4K230, in several human tissues, including brain, suggests that these enzymes are involved in distinct basic cellular functions.

The distribution of tissue fibronectin and sialic acid in human breast cancer.

Our findings indicate that sialic acid and fibronectin levels in breast tumors are higher than those in normal tissues. The mean tissue fibronectin and sialic acid concentrations for patients with breast cancer were 30.90 +/- 9.68 microg/mg protein and 21.60 +/- 9.35 microg/mg protein, respectively, and for normal controls were 12.47 +/- 5.69 microg/mg protein, respectively. Tissue fibronectin and sialic acid can be important markers for human breast cancer.

The importance of Lp(a)-fibronectin interaction in atherogenesis.

An elevated concentration of lipoprotein (a) (Lp(a)) in serum has been considered a risk factor for coronary heart disease by various investigators. The apo(a) portion of Lp(a) binds to the carboxyterminal heparin binding domain of fibronectin. Lp(a) bound to fibronectin is internalized through the fibronectin receptor pathway and thereby causes increased accumulation of lipid and foam cell formation. In the present study, fibronectin and Lp(a) concentrations have been assayed in patients with coronary heart disease (CHD) and healthy subjects. Serum total cholesterol, triglyceride, LDL, VLDL and HDL cholesterol levels in patients with coronary heart disease were significantly different from the control group (p < 0.001). Plasma fibronectin (p < 0.05) and serum Lp(a) (p < 0.001) levels were found to be significantly elevated when compared with the control group.

The molecular markers of hemostatic activation on coronary artery disease.

Endothelial cells, circulating platelets, and proteins of the coagulation and fibrinolytic systems are known to contribute to the hemostatic processes. Various molecular markers of hemostatic alteration are found in increased amounts in the circulation during the activation of this process. In this study, we investigated serum lipoprotein (a) and plasma platelet factor 4, beta-thromboglobulin, thrombin-anthithrombin complex, fibrinopeptid A, D-dimer, tissue plasminogen activator, tissue plasminogen activator inhibitor, and fibronectin levels in patients with coronary artery disease. The levels of all these markers were found to be significantly higher as compared to the control group. Our findings suggest that patients with coronary artery disease have greater blood coagulability than controls, and the use of molecular markers has become greatly important in clinical practice.

Plasma Lp(a) and t-PA-PAI-1 complex levels in coronary heart disease.

In this study we investigated serum Lp(a) and plasma t-PA-PAI-1 complex levels in patients with coronary heart disease (CHD). Serum total cholesterol, triglyceride, LDL and VDL cholesterol levels (p < 0.001) and HDL cholesterol levels (p < 0.01) in patients group were found to be significantly different from those in control group. The mean Lp(a) and t-PA-PAI-1 complex levels in patients with coronary heart disease were significantly higher as compared to control group (p < 0.001). This data indicate that the elevated levels of serum Lp(a) and plasma t-PA-PAI-1 complex may play an important role in the pathogenesis of coronary atherosclerosis.

The prognostic importance of fibronectin and sialic acid levels in human pituitary adenomas.

In this study, fibronectin and sialic acid levels were determined in human pituitary adenomas. The mean fibronectin and sialic acid levels for human pituitary adenomas were found to be 31.64 +/- 15.82 microgram/mg protein and 21.90 +/- 9.82 microgram/mg protein, respectively, versus 6.30 +/- 2.96 microgram/mg protein and 9.88 +/- 2.81 microgram/mg protein for the normal brain tissues. Fibronectin and sialic acid levels were significantly higher (P < 0.001) in human pituitary adenomas than the normal brain tissues. In human infiltrative and non-infiltrative pituitary adenomas, the mean fibronectin and sialic acid levels were found to be 40.87 +/- 15.90 microgram/mg protein, 27.59 +/- 11.10 microgram/mg protein and 22.40 +/- 9.51 microgram/mg protein, 16.21 +/- 3.20 microgram/mg protein, respectively. Fibronectin and sialic acid levels were slightly elevated (P < 0.05) in human infiltrative pituitary adenomas compared with non-infiltrative adenomas.

Tissue sialic acid and fibronectin levels in human prostatic cancer.

We investigated the tissue concentration of sialic acid and fibronectin in patients with prostatic cancer. The mean sialic acid and fibronectin levels in patients with prostatic cancer were 19.02 +/- 6.30 micrograms/mg protein, respectively versus 13.01 +/- 4.53 micrograms/mg protein and 11.77 +/- 6.74 micrograms/mg protein for normal prostatic tissues. Sialic acid and fibronectin levels in cancerous patients were significantly higher than in the control group (P < 0.05).

The relationship between the electrophoretic mobility of lipoproteins and coronary heart disease.

Low density lipoproteins (LDL) in patients with coronary atherosclerosis have a substantially lower content of sialic acid when compared with the LDL from healthy subjects. Desialylated LDL have smaller sizes and greater electrophoretic mobilities than sialylated ones. Desialylated LDL may be responsible for the accelerated development of foam cells in atherosclerosis. In the present study, we investigated a relationship between the electrophoretic mobility of lipoproteins and the number of significantly obstructed vessels in patients with coronary heart disease (CHD). Our findings indicate that when the number of significantly obstructed vessels is increased, the electrophoretic mobility of lipoproteins is high. We also investigated the possible role of serum sialidase activity on lipoprotein desialylation in patients with coronary heart disease. In patients with single vessel disease (p < 0.01) and double-triple vessel disease (p < 0.001) the mean serum sialidase activity was significantly higher than in the control group.

Tissue fibronectin levels of human prostatic cancer, as a tumor marker.

In this study, tissue fibronectin levels have been assayed in human prostatic cancer, benign prostatic hyperplasia and normal prostatic tissue. The mean tissue fibronectin levels for prostatic cancer, benign prostatic hyperplasia and normal groups were found to be 20.22 +/- 8.66 micrograms/mg protein and 11.77 +/- 6.74 micrograms/mg protein, respectively. In the malignant group, the mean fibronectin concentrations, appeared to be significantly higher than normal, (p < 0.05). On the other hand, fibronectin levels of benign prostatic hyperplasia were found to be statistically insignificant in comparison to the normal group (p > 0.05).

Plasma fibronectin and sialic acid levels in various types of human brain tumors.

In this study, fibronectin and sialic acid concentrations were determined in plasma from patients with pituitary adenoma, meningioma and glioma, and, from controls. The mean plasma fibronectin levels in patients with pituitary adenoma, meningioma and glioma (p < 0.001) appeared to be significantly lower than controls. On the contrary, the mean plasma sialic acid values in patients with pituitary adenoma (p < 0.01), and glioma (p < 0.001) were significantly higher as compared to normal plasmas. The mean plasma sialic acid values in patients with meningioma were not different from those in controls. Also, the mean fibronectin levels in patients with glioma were found to be significantly higher than those in patients with meningioma (p < 0.05).

Fibronectin and sialic acid levels in human meningiomas and gliomas.

In this study, fibronectin and sialic acid levels have been assayed in human meningiomas and gliomas. The mean fibronectin and sialic acid levels for human meningiomas were 22.01 +/- 9.70 micrograms/mg protein and 19.58 +/- 4.89 micrograms/mg protein, respectively, and for human gliomas were 27.30 +/- 13.70 micrograms/mg protein and 25.67 +/- 11.60 micrograms/mg protein, respectively, versus 9.23 +/- 5.40 micrograms/mg protein and 13.50 +/- 4.30 micrograms/mg protein for normal brain tissues. Fibronectin and sialic acid levels were significantly higher in human meningiomas (P < 0.01 and P < 0.05) and gliomas (P < 0.001 and P < 0.01) than control group. Also the mean fibronectin and sialic acid levels were found to be 18.27 +/- 7.08 micrograms/mg protein and 17.04 +/- 6.25 micrograms/mg protein in Grade I-II and 32.60 +/- 15.00 micrograms/mg protein and 29.50 +/- 11.60 micrograms/mg protein in Grade III-IV gliomas, respectively. Fibronectin and sialic acid levels were significantly higher in Grade III-IV gliomas than Grade I-II gliomas (P < 0.05).

Relation between 22-kHz ultrasonic signals and sociosexual behavior in rats.

The copulatory performance of male rats, tested in a large seminaturalistic environment, was assessed to determine the relation between 22-kHz ultrasonic vocalizations and a range of sociosexual behaviors. The male rats were tested until sexual exhaustion. Such ultrasonic signals were shown to occur in a wider range of sociosexual circumstances than previously reported; for example, the calls occcurred in particular social circumstances during the preejaculatory period as well as during the postejaculatory interval. There was no consistent evidence that the emission of this call during the postejaculatory period consistently functions to keep the female away from the male. The nature and occurrence of postejaculatory ultrasonic signals showed increasing variability in successive ejaculatory series. The results of this and previous studies are interpreted within a semiotic theory of communication. The 22-kHz call is described as a message that makes available the information that the sender is in a socially depressed and withdrawn state.

Evaluation of the Swank IL-201 transfusion filter.

The effects of a new transfusion filter (Swank IL-20U) on stored, whole blood have been examined. Six filters were preloaded by passage of two units of outdated, type-specific bank blood, and the effects of filtration on a third unit of 21-day-old blood flowing under 150 mm Hg pressure were measured. Filtration did not significantly alter erythrocyte or leukocyte count, total or plasma hemoglobin, red-cell fragility, and plasma sodium, potassium, albumin, or globulin. Platelet counts were reduced by 33%. Removal of microaggregates, assessed by Coulter counting, screen filtration pressure, total screen protein, wet and dry weight of material retained, and scanning electron microscopy, was shown to be effective over the entire range of particle size. In comparison with other transfusion filters previously examined in this laboratory, the Swank IL-201 filter combines the features of efficient microaggregate removal with moderate blood-flow rate. Compared to its predecessor, the Swank IL-200, this new filter design has improved flow characteristics without loss of microaggregate removal efficiency. In view of the similarity of performance demonstrated for several of the available filters, it seems likely that relative cost will constitute an important determinant of filter selection.