A major ozonation product of cholesterol, 3beta-hydroxy-5-oxo-5,6-secocholestan-6-al, induces apoptosis in H9c2 cardiomyoblasts

Cholesterol, a major neutral lipid component of biological membranes and the lung epithelial lining fluids, is susceptible to oxidation by reactive oxygen and nitrogen species including ozone. The oxidation by ozone in biological environments results in the formation of 3beta-hydroxy-5-oxo-5,6-secocholestan-6-al (cholesterol secoaldehyde or CSeco, major product) along with some other minor products. Recently, CSeco has been implicated in the pathogenesis of atherosclerosis and Alzheimer's disease. In this communication, we report that CSeco induces cytotoxicity in H9c2 cardiomyoblasts with an IC(50) of 8.9+/-1.29 microM (n=6). The observed effect of CSeco at low micromolar concentrations retained several key features of apoptosis, such as changes in nuclear morphology, phosphatidylserine externalization, DNA fragmentation, and caspase 3/7 activity. Treatment of cardiomyocytes with 5 microM CSeco for 24h, for instance, resulted in 30.8+/-3.28% apoptotic and 1.8+/-1.11% of necrotic cells as against DMSO controls that only showed 1.3+/-0.33% of apoptosis and 1.6+/-0.67% of necrosis. In general, the loss of cellular viability paralleled the increased occurrence of apoptotic cells in various CSeco treatments. This study, for the first time, demonstrates the induction of apoptotic cell death in cardiomyocytes by a cholesterol ozonation product, implying a role for ozone in myocardial injury.     

Critical balance of electrostatic and hydrophobic interactions is required for beta 2-microglobulin amyloid fibril growth and stability

Investigation of factors that modulate amyloid formation of proteins is important to understand and mitigate amyloid-related diseases. To understand the role of electrostatic interactions and the effect of ionic cosolutes, especially anions, on amyloid formation, we have investigated the effect of salts such as NaCl, NaI, NaClO(4), and Na(2)SO(4) on the amyloid fibril growth of beta(2)-microglobulin, the protein involved in dialysis-related amyloidosis. Under acidic conditions, these salts exhibit characteristic optimal concentrations where the fibril growth is favored. The presence of salts leads to an increase in hydrophobicity of the protein as reported by 8-anilinonaphthalene-1-sulfonic acid, indicating that the anion interaction leads to the necessary electrostatic and hydrophobic balance critical for amyloid formation. However, high concentrations of salts tilt the balance to high hydrophobicity, leading to partitioning of the protein to amorphous aggregates. Such amorphous aggregates are not competent for fibril growth. The order of anions based on the lowest concentration at which fibril formation is favored is SO(4)(2)(-) > ClO(4)(-) > I(-) > Cl(-), consistent with the order of their electroselectivity series, suggesting that preferential anion binding, rather than general ionic strength effect, plays an important role in the amyloid fibril growth. Anion binding is also found to stabilize the amyloid fibrils under acidic condition. Interestingly, sulfate promotes amyloid growth of beta(2)-microglobulin at pH between 5 and 6, closer to its isoelectric point. Considering the earlier studies on the role of glycosaminoglycans and proteoglycans (i.e., sulfated polyanions) on amyloid formation, our study suggests that preferential interaction of sulfate ions with amyloidogenic proteins may have biological significance.     

Attempts to delineate the relative contributions of changes in hydrophobicity and packing to changes in stability of ribonuclease S mutants

While the hydrophobic driving force is thought to be a major contributor to protein stability, it is difficult to experimentally dissect out its contribution to the overall free energy of folding. We have made large to small substitutions of buried hydrophobic residues at positions 8 and 13 in the peptide/protein complex, RNase-S, and have characterized the structures by X-ray crystallography. The thermodynamics of association of these mutant S peptides with S protein was measured in the presence of different concentrations of methanol and ethanol. The reduction in the strength of the hydrophobic driving force in the presence of these organic solvents was estimated from surface-tension data as well as from the dependence of the DeltaC(p) of protein/peptide binding on the alcohol concentration. The data indicated a decrease in the strength of the hydrophobic driving force of about 30-40% over a 0-30% range of the alcohol concentration. We observe that large to small substitutions destabilize the protein. However, the amount of destabilization, relative to the wild type, is independent of the alcohol concentration over the range of alcohol concentrations studied. The data clearly indicate that decreased stability of the mutants is primarily due to the loss of packing interactions rather than a reduced hydrophobic driving force and suggest a value of the hydrophobic driving force of less than 18 cal mol(-)(1) A(2).     

Exogenous AdoMet and its analogue sinefungin differentially influence DNA cleavage by R.EcoP15I--usefulness in SAGE

While it has been demonstrated that AdoMet is required for DNA cleavage by Type III restriction enzymes, here we show that in the presence of exogenous AdoMet, the head-to-head oriented recognition sites are cleaved only on a supercoiled DNA. On a linear DNA, exogenous AdoMet strongly drives methylation while inhibiting cleavage reaction. Strikingly, AdoMet analogue sinefungin results in cleavage at all recognition sites irrespective of the topology of DNA. The cleavage reaction in the presence of sinefungin is ATP dependent. The site of cleavage is comparable with that in the presence of AdoMet. The use of EcoP15I restriction in presence of sinefungin as an improved tool for serial analysis of gene expression is discussed.     

Discovery of a novel superfamily of type III polyketide synthases in Aspergillus oryzae

Identification of genes encoding type III polyketide synthase (PKS) superfamily members in the industrially useful filamentous fungus, Aspergillus oryzae, revealed that their distribution is not specific to plants or bacteria. Among other Aspergilli (Aspergillus nidulans and Aspergillus fumigatus), A. oryzae was unique in possessing four chalcone synthase (CHS)-like genes (csyA, csyB, csyC, and csyD). Expression of csyA, csyB, and csyD genes was confirmed by RT-PCR. Comparative genome analyses revealed single putative type III PKS in Neurospora crassa and Fusarium graminearum, two each in Magnaporthe grisea and Podospora anserina, and three in Phenarocheate chrysosporium, with a phylogenic distinction from bacteria and plants. Conservation of catalytic residues in the CHSs across species implicated enzymatically active nature of these newly discovered homologs.     

Three-dimensional image analysis of plugging at the septal pore by Woronin body during hypotonic shock inducing hyphal tip bursting in the filamentous fungus Aspergillus oryzae

We observed that the filamentous fungus, Aspergillus oryzae, grown on agar media burst out cytoplasmic constituents from the hyphal tip soon after flooding with water. Woronin body is a specialized organelle known to plug the septal pore adjacent to the lysed compartment to prevent extensive loss of cytoplasm. A. oryzae Aohex1 gene homologous to Neurospora crassa HEX1 gene encoding a major protein in Woronin body was expressed as a fusion with DsRed2, resulting in visualization of Woronin body. Confocal microscopy and three-dimensional reconstruction of images visualized the septal pore as a dark region surrounded by green fluorescence of EGFP-fused secretory protein, RNase T1, on the septum. Dual fluorescent labeling revealed the plugging of the septal pores adjacent to the lysed apical compartments by Woronin bodies during hypotonic shock. Disruption of Aohex1 gene caused disappearance of Woronin bodies and the defect to prevent extensive loss of cytoplasm during hypotonic shock.     

The catalytically active serine protease domain of human complement factor I

Factor I (fI) is a major regulator of complement. As a protease it has very restricted specificity, cleaving only C3b or C4b in the presence of a cofactor such as factor H (fH). Cleavage of C3b by fI yields iC3b, a major opsonin. The cleavage occurs through the formation of a ternary complex between the enzyme, the substrate, and the cofactor. The catalytic subunit of fI, the SP domain, accommodates substrate recognition and cleavage. The role of the fI heavy chain within the catalysis complex is unknown. Using partial proteolysis and affinity chromatography an intact form of the SP domain was generated and isolated from fI in high yield. fI and the SP domain were found to have similar amidolytic activities but strikingly different proteolytic activities on C3(NH(3)). fI did not cleave C3(NH(3)) in the absence of fH, while in its presence it cleaved C3(NH(3)) rapidly at two sites. The SP domain, however, slowly cleaved C3(NH(3)) in the absence of fH, at more than two sites. Cleavage by the SP domain was inhibited, not stimulated, by fH. Pefabloc SC and antipain inhibited the proteolytic activity of both fI and the SP domain, but suramin inhibited only fI and not the SP domain. The contrast in the proteolytic activities suggests that the heavy chain domains and the cofactor must have roles in orienting the natural substrates and restricting cleavage to the two sites which yield iC3b through a highly specific catalysis.     

Common genetic and environmental effects on lipid phenotypes: the HERITAGE family study

OBJECTIVE: Despite the well known genetic component influencing plasma lipid-lipoprotein levels and the observed correlations among these traits, little is known about pleiotropic heritable determinants among them. Our aim is to investigate pair-wise polygenic and environmental correlations among lipid-lipoprotein levels at baseline and in response to regular exercise in Whites and Blacks. METHODS: Common pair-wise genetic and environmental correlations among levels of total cholesterol (TC), LDL-C, ApoB, HDL-C (also HDL2-C and HDL3-C), triglycerides (TG, HDL-TG and LDL-TG) and ApoA-1 were investigated at baseline and again after a 20-week endurance exercise program using a variance-components-decomposition. RESULTS: With a few exceptions, all lipid phenotypes were heritable at baseline and for training responses in Blacks and Whites. Strong to high genetic and environmental correlations (0.4 < rho(g) < 0.7) were observed for the majority of the baseline pair-wise traits. For training responses, many of the same patterns were noted, although fewer genetic correlations were significant as compared to the baseline results. CONCLUSIONS: Results suggest that the observed phenotypic correlations among many of these traits may be due to in part to pleiotropic genes, in particular between LDL-C and ApoB and between TG and HDL-C. This shared genetic architecture should be considered in follow-up gene finding studies.     

Solitary fibrous tumor: report of a case with an unusual presentation as a spindle cell parotid neoplasm

BACKGROUND: Initially described as a pleural tumor, solitaryfibrous tumor of the parotid gland (SFT) is rare and has been reported at a wide range ofanatomic sites. Although cases of SFT arising in the parotid gland have been previously described, a review of the literature failed to reveal cytology-based reports of this entity. CASE: A 42-year-old man presented with a right parotid mass that had gradually enlarged over 3 years. He was otherwise asymptomatic. Fine needle aspiration biopsy of the mass showed a hypercellular smear composed of spindle cells in both clusters and isolated forms, with ovoid nuclei, evenly distributed chromatin, inconspicuous nucleoli and scant to moderate cytoplasm with focally wispy, collagenous, intercellular material. The background was hemorrhagic, without chondromyxoid matrix or inflammatory cells. There was no evidence of a myoepithelial component. A diagnosis of spindle cell neoplasm was rendered. Histologic examination of the total parotidectomy specimen revealed a SFT arising in the parotid gland. The diagnosis was supported by immunohistochemical studies. CONCLUSION: SFT is a well-circumscribed neoplasm composed of short, spindled, plump cells with scanty cytoplasm growing in a haphazard or "patternless" pattern. Tumor cells are intimately admixed with collagenous stroma. Hemangiopericytomalike vessels are frequently seen. Although SFT rarely occurs in the salivary gland and a definitive diagnosis based on cytologic preparations alone is difficult, the diagnosis of SFT can be considered when cytologic examination reveals a hypercellular smear composed of isolated, cohesive clusters of spindled, fibroblastlike cells associated with a collagenous component in ahemorrhagic background. The preoperative magnetic esonance image findings of a highly vascular neoplasm support the diagnosis.