Hyperphosphorylation of extracellular regulated kinase 2 (ERK2) and inhibition of JNK2 phosphorylation are associated with increased S-phase during transformation of Syrian hamster embryo cells by Malachite Green

Malachite Green (MG), consisting of green crystals with a metallic lustre, is highly soluble in water, cytotoxic to various mammalian cells and also acts as a liver tumour promoter. In view of its industrial importance and possible exposure to human beings, MG poses a potential environmental health hazard. We have earlier reported the malignant transformation of Syrian hamster embryo (SHE) cells in primary culture by MG. In this study, we have studied the mitogen activated protein (MAP) kinase signal transduction pathway in preneoplastic cells induced by MG. Western blots of MG induced preneoplastic cells showed no phosphorylation of ERK1, an increased phosphoactive ERK2 associated with a decreased expression of phosphoactive JNK2. However, total forms of ERKs, JNKs and p38 Kinases showed similar levels of expression in control and preneoplastic SHE cells. Indirect immunofluorescence studies have shown a distinct nuclear localisation of phosphoactive ERKs in MG induced preneoplastic cells. Flow cytometric analysis showed an increase of S-phase cells in preneoplastic cells compared to control SHE cells. The present study indicates that hyperphosphorylation of ERK2, decreased JNK2 phosphorylation and an increase in S-phase cells seems to be the early changes associated with the MG induced malignant transformation of SHE cells in primary culture.     

Atomic resolution analysis of the catalytic site of an aspartic proteinase and an unexpected mode of binding by short peptides

The X-ray structures of native endothiapepsin and a complex with a hydroxyethylene transition state analog inhibitor (H261) have been determined at atomic resolution. Unrestrained refinement of the carboxyl groups of the enzyme by using the atomic resolution data indicates that both catalytic aspartates in the native enzyme share a single negative charge equally; that is, in the crystal, one half of the active sites have Asp 32 ionized and the other half have Asp 215 ionized. The electron density map of the native enzyme refined at 0.9 A resolution demonstrates that there is a short peptide (probably Ser-Thr) bound noncovalently in the active site cleft. The N-terminal nitrogen of the dipeptide interacts with the aspartate diad of the enzyme by hydrogen bonds involving the carboxyl of Asp 215 and the catalytic water molecule. This is consistent with classical findings that the aspartic proteinases can be inhibited weakly by short peptides and that these enzymes can catalyze transpeptidation reactions. The dipeptide may originate from autolysis of the N-terminal Ser-Thr sequence of the enzyme during crystallization.     

Differential distribution of the cognate and heat-stress-induced isoforms of high Mr cis-trans prolyl peptidyl isomerase (FKBP) in the cytoplasm and nucleoplasm

Wheat root tips express a 73 kDa cognate isoform and a 77 kDa heat-shock-induced isoform of peptidyl prolyl cis-trans isomerase (FK506 binding protein; FKBP) that is part of a chaperone complex with hsp90. The 73 kDa and 77 kDa FKBPs have very similar sequences, differing primarily in the N- and C-terminal 20 amino acids. In order to define the potential functional roles of these proteins, the 73 kDa and 77 kDa FKBPs were localized in root tips using antigen-affinity purified antibodies as a probe. The cognate 73 kDa FKBP is localized in the cytoplasm and appears enriched around the periphery of the early vacuole and vesicles exiting the trans-Golgi. Parallel assays with antibodies directed against tonoplast aquaporin and pyrophosphatase confirmed the association of FKBP with an early vacuole compartment. Sucrose gradient centrifugation analysis of root tip lysates also showed that 73 kDa FKBP is co-fractionated with tonoplast aquaporin and V-ATPase in a light compartment near the top of the gradient. Heat-shock treatment of root tips induces the accumulation of 77 kDa FKBP while the abundance of 73 kDa FKBP remains constant. Quantitative EM immunogold assays of the intracellular distribution of FKBP over an 8 h heat-shock time-course showed that FKBP is initially present in the cytoplasm, but is transported into the nucleus where it accumulates in the nucleoplasm and into specific subnuclear domains. The results of this study show that the intracellular distribution of the high Mr FKBPs in wheat root tips differs at normal and elevated temperatures, indicating different functional roles for the FKBP isoforms.     

Uniformly minimum variance unbiased estimation of gene diversity

Gene diversity is an important measure of genetic variability in inbred populations. The survival of species in changing environments depends on, among other factors, the genetic variability of the population. In this communication, I have derived the uniformly minimum variance unbiased estimator of gene diversity. The proposed estimator of gene diversity does not assume that the inbreeding coefficient is known. I have also provided the approximate variance of this estimator according to Fisher's method. In addition, I have developed a numerical resampling-based method for obtaining variances and confidence intervals based on the maximum likelihood estimator and the uniformly minimum variance unbiased estimator. Efficiency in estimation of the gene diversity based on these two estimators is discussed. In accordance with the simulation results, I found that the uniformly minimum variance estimator developed in this report is more accurate for estimation of gene diversity than the maximum likelihood estimator.     

Innate immunity mediated by the cytokine IL-1 homologue 4 (IL-1H4/IL-1F7) induces IL-12-dependent adaptive and profound antitumor immunity

Recently, several novel members of the IL-1 family have been identified. The possible therapeutic utility and the underlying biologic role of these new members remain unclear. In the present study we analyzed the anti-tumor activity of human IL-1 homologue 4(IL-1H4; renamed IL-F7) by adenovirus-mediated gene transfer (AdIL-1H4) directly into murine tumors. In vitro expression analysis showed that IL-1H4 was a secretory protein. Treatment of an established MCA205 mouse fibrosarcoma by single intratumoral injection of AdIL-1H4 resulted in significant growth suppression. Furthermore, complete inhibition of tumor growth was observed following multiple injections of AdIL-1H4. The anti-tumor activity of IL-1H4 was abrogated in nude and SCID mice and in IL-12-, IFN-gamma-, or Fas ligand-deficient mice. In contrast, IL-1H4 was able to confer substantial anti-tumor effects in NKT-deficient mice. These results suggest that IL-1H4 could play an important role in the link between innate and adaptive immunity and may be useful for tumor immunotherapy.     

The ATP binding cassette multidrug transporter LmrA and lipid transporter MsbA have overlapping substrate specificities

LmrA is an ATP binding cassette (ABC) multidrug transporter in Lactococcus lactis that is a structural and functional homologue of the human multidrug resistance P-glycoprotein MDR1 (ABCB1). LmrA is also homologous to MsbA, an essential ABC transporter in Escherichia coli involved in the trafficking of lipids, including Lipid A. We have compared the substrate specificities of LmrA and MsbA in detail. Surprisingly, LmrA was able to functionally substitute for a temperature-sensitive mutant MsbA in E. coli WD2 at non-permissive temperatures, suggesting that LmrA could transport Lipid A. LmrA also exhibited a Lipid A-stimulated, vanadate-sensitive ATPase activity. Reciprocally, the expression of MsbA conferred multidrug resistance on E. coli. Similar to LmrA, MsbA interacted with photoactivatable substrate [3H]azidopine, displayed a daunomycin, vinblastine, and Hoechst 33342-stimulated vanadate-sensitive ATPase activity, and mediated the transport of ethidium from cells and Hoechst 33342 in proteoliposomes containing purified and functionally reconstituted protein. Taken together, these data demonstrate that MsbA and LmrA have overlapping substrate specificities. Our observations imply the presence of structural elements in the recently published crystal structures of MsbA in E. coli and Vibrio cholera (Chang, G., and Roth, C. B. (2001) Science 293, 1793-1800; Chang, G. (2003) J. Mol. Biol. 330, 419-430) that support drug-protein interactions and suggest a possible role for LmrA in lipid trafficking in L. lactis.     

Extracellular hyaline material in association with other cytologic features in aspirates from collagenous spherulosis and adenoid cystic carcinoma of the breast

OBJECTIVE: To reevaluate breast aspirates showing extracellular hyaline material (EHM) and globules to assess if clinicoradiologic and cytologic features could help in differentiating between benign and malignant lesions, especially collagenous spherulosis (CS) and adenoid cystic carcinoma (ACC). STUDY DESIGN: Fine needle aspiration was performed on 884 patients with breast lumps. The cytomorphologic features of 6 cases showing EHM, including classic hyaline globules (HGs), were analyzed in detail. Three cases also had hemorrhagic nipple discharge. Tissue diagnosis (4) and mammography (6) were available. RESULTS: Aspirate smears revealed high cellularity composed of monolayers: clusters of uniform, small cells; EHM; and HGs surrounded by similar cells. Benign naked nuclei and stromal fragments (4), nuclear pleomorphism (3), apocrine cells (2), foam cells (2), naked HGs (2) and spindle cells in proximity to HGs were also seen (4). Nipple discharge smears showed foam cells, erythrocytes (3) and epithelial cell clusters with hyaline material (1). The cytologic diagnosis was CS (4) and ACC (2). Histopathology confirmed the diagnosis of CS (2) and ACC (1). CONCLUSION: There may be a morphologic overlap between the cytomorphologic features of CS and ACC, leading to diagnostic errors. The presence of EHM and HGs in association with bland cellular features should be interpreted with caution to avoid erroneous diagnoses. Histopathology is mandatory in these cases because of their different prognostic implications.     

A whole-genome scan for obstructive sleep apnea and obesity

Obstructive sleep apnea (OSA) is a common, chronic, complex disease associated with serious cardiovascular and neuropsychological sequelae and with substantial social and economic costs. Along with male gender, obesity is the most characteristic feature of OSA in adults. To identify susceptibility loci for OSA, we undertook a 9-cM genome scan in 66 white pedigrees (n=349 subjects) ascertained on the basis of either an affected individual with laboratory-confirmed OSA or a proband who was a neighborhood control individual. Multipoint variance-component linkage analysis was performed for the OSA-associated quantitative phenotypes apnea-hypopnea index (AHI) and body mass index (BMI). Candidate regions on chromosomes 1p (LOD score 1.39), 2p (LOD score 1.64), 12p (LOD score 1.43), and 19p (LOD score 1.40) gave the most evidence for linkage to AHI. BMI was also linked to multiple regions, most significantly to markers on chromosomes 2p (LOD score 3.08), 7p (LOD score 2.53), and 12p (LOD score 3.41). Extended modeling indicated that the evidence for linkage to AHI was effectively removed after adjustment for BMI, with the exception of the candidate regions on chromosomes 2p (adjusted LOD score 1.33) and 19p (adjusted LOD score 1.45). After adjustment for AHI, the primary linkages to BMI remained suggestive but were roughly halved. Our results suggest that there are both shared and unshared genetic factors underlying susceptibility to OSA and obesity and that the interrelationship of OSA and obesity in white individuals may be partially explained by a common causal pathway involving one or more genes regulating both AHI and BMI levels.     

Molecular pathway for (-)-epigallocatechin-3-gallate-induced cell cycle arrest and apoptosis of human prostate carcinoma cells

Epigallocatechin-3-gallate (EGCG), the major polyphenolic constituent present in green tea, is a promising chemopreventive agent. We recently showed that green tea polyphenols exert remarkable preventive effects against prostate cancer in a mouse model and many of these effects are mediated by the ability of polyphenols to induce apoptosis in cancer cells [Proc. Natl. Acad. Sci. USA 98 (2001) 10350]. Earlier, we showed that EGCG causes a G0/G1 phase cell cycle arrest and apoptosis of both androgen-sensitive LNCaP and androgen-insensitive DU145 human prostate carcinoma cells, irrespective of p53 status [Toxicol. Appl. Pharmacol. 164 (2000) 82]. Here, we provide molecular understanding of this effect. We tested a hypothesis that EGCG-mediated cell cycle dysregulation and apoptosis is mediated via modulation of cyclin kinase inhibitor (cki)-cyclin-cyclin-dependent kinase (cdk) machinery. As shown by immunoblot analysis, EGCG treatment of LNCaP and DU145 cells resulted in significant dose- and time-dependent (i) upregulation of the protein expression of WAF1/p21, KIP1/p27, INK4a/p16, and INK4c/p18, (ii) down-modulation of the protein expression of cyclin D1, cyclin E, cdk2, cdk4, and cdk6, but not of cyclin D2, (iii) increase in the binding of cyclin D1 toward WAF1/p21 and KIP1/p27, and (iv) decrease in the binding of cyclin E toward cdk2. Taken together, our results suggest that EGCG causes an induction of G1 phase ckis, which inhibits the cyclin-cdk complexes operative in the G0/G1 phase of the cell cycle, thereby causing an arrest, which may be an irreversible process ultimately leading to apoptotic cell death. This is the first systematic study showing the involvement of each component of cdk inhibitor-cyclin-cdk machinery during cell cycle arrest and apoptosis of human prostate carcinoma cells by EGCG.