Up-Regulation of Hepatoma-Derived Growth Factor Facilities Tumor Progression in Malignant Melanoma

Cutaneous malignant melanoma is the fastest increasing malignancy in humans. Hepatoma-derived growth factor (HDGF) is a novel growth factor identified from human hepatoma cell line. HDGF overexpression is correlated with poor prognosis in various types of cancer including melanoma. However, the underlying mechanism of HDGF overexpression in developing melanoma remains unclear. In this study, human melanoma cell lines (A375, A2058, MEL-RM and MM200) showed higher levels of HDGF gene expression, whereas human epidermal melanocytes (HEMn) expressed less. Exogenous application of HDGF stimulated colony formation and invasion of human melanoma cells. Moreover, HDGF overexpression stimulated the degree of invasion and colony formation of B16–F10 melanoma cells whereas HDGF knockdown exerted opposite effects in vitro. To evaluate the effects of HDGF on tumour growth and metastasis in vivo, syngeneic mouse melanoma and metastatic melanoma models were performed by manipulating the gene expression of HDGF in melanoma cells. It was found that mice injected with HDGF-overexpressing melanoma cells had greater tumour growth and higher metastatic capability. In contrast, mice implanted with HDGF-depleted melanoma cells exhibited reduced tumor burden and lung metastasis. Histological analysis of excised tumors revealed higher degree of cell proliferation and neovascularization in HDGF-overexpressing melanoma. The present study provides evidence that HDGF promotes tumor progression of melanoma and targeting HDGF may constitute a novel strategy for the treatment of melanoma.     

Pentaketide melanin biosynthesis in Aspergillus fumigatus requires chain-length shortening of a heptaketide precursor

Chain lengths and cyclization patterns of microbial polyketides are generally determined by polyketide synthases alone. Fungal polyketide melanins are often derived from a pentaketide 1,8-dihydroxynaphthalene, and pentaketide synthases are used for synthesis of the upstream pentaketide precursor, 1,3,6,8-tetrahydroxynaphthalene (1,3,6,8-THN). However, Aspergillus fumigatus, a human fungal pathogen, uses a heptaketide synthase (Alb1p) to synthesize its conidial pigment through a pentaketide pathway similar to that which produces 1,8-dihydroxynaphthalene-melanin. In this study we demonstrate that a novel protein, Ayg1p, is involved in the formation of 1,3,6,8-THN by chain-length shortening of a heptaketide precursor in A. fumigatus. Deletion of the ayg1 gene prevented the accumulation of 1,3,6,8-THN suggesting the involvement of ayg1 in 1,3,6,8-THN production. Genetic analyses of double-gene deletants suggested that Ayg1p catalyzes a novel biosynthetic step downstream of Alb1p and upstream of Arp2p (1,3,6,8-THN reductase). Further genetic and biochemical analyses of the reconstituted strains carrying alb1, ayg1, or alb1 + ayg1 indicated that Ayg1p is essential for synthesis of 1,3,6,8-THN in addition to Alb1p. Cell-free enzyme assays, using the crude Ayg1p protein extract, revealed that Ayg1p enzymatically shortened the heptaketide product of Alb1p to 1,3,6,8-THN. Thus, the protein Ayg1p facilitates the participation of a heptaketide synthase in a pentaketide pathway via a novel polyketide-shortening mechanism in A. fumigatus.     

Multifunctionality of liver alcohol dehydrogenase: Kinetic and mechanistic studies of esterase reaction

Alcohol dehydrogenase from horse liver is shown to catalyze ester hydrolysis. Nicotinamide coenzymes do not affect the rate of esterolysis. A kinetic approach to study esterase reaction at low substrate to enzyme ratio is described. Kinetic effects of ester structure, temperature, pH, solvent polarity, and ionic strength were investigated. The liver enzyme enhances the rate of esterolysis by lowering activation energy of reaction according to the Uni-Bi kinetic sequence. Two ionizable groups, cysteine and lysine, are tentatively assigned at the esterolytic site of liver alcohol dehydrogenase from pH-rate profiles and chemical modification studies. A plausible mechanism for the esterase reaction proceeds via the acid-assisted nucleophilic catalysis involving the ammonium ion of lysine and the thiolate of cysteine in the acyl-oxygen cleavage.     

Stress-induced phosphorylation and proteasomal degradation of mitofusin 2 facilitates mitochondrial fragmentation and apoptosis

Mitochondria play central roles in integrating pro- and antiapoptotic stimuli, and JNK is well known to have roles in activating apoptotic pathways. We establish a critical link between stress-induced JNK activation, mitofusin 2, which is an essential component of the mitochondrial outer membrane fusion apparatus, and the ubiquitin-proteasome system (UPS). JNK phosphorylation of mitofusin 2 in response to cellular stress leads to recruitment of the ubiquitin ligase (E3) Huwe1/Mule/ARF-BP1/HectH9/E3Histone/Lasu1 to mitofusin 2, with the BH3 domain of Huwe1 implicated in this interaction. This results in ubiquitin-mediated proteasomal degradation of mitofusin 2, leading to mitochondrial fragmentation and enhanced apoptotic cell death. The stability of a nonphosphorylatable mitofusin 2 mutant is unaffected by stress and protective against apoptosis. Conversely, a mitofusin 2 phosphomimic is more rapidly degraded without cellular stress. These findings demonstrate how proximal signaling events can influence both mitochondrial dynamics and apoptosis through phosphorylation-stimulated degradation of the mitochondrial fusion machinery.     

Protein identification using top-down

In the last two years, because of advances in protein separation and mass spectrometry, top-down mass spectrometry moved from analyzing single proteins to analyzing complex samples and identifying hundreds and even thousands of proteins. However, computational tools for database search of top-down spectra against protein databases are still in their infancy. We describe MS-Align+, a fast algorithm for top-down protein identification based on spectral alignment that enables searches for unexpected post-translational modifications. We also propose a method for evaluating statistical significance of top-down protein identifications and further benchmark various software tools on two top-down data sets from Saccharomyces cerevisiae and Salmonella typhimurium. We demonstrate that MS-Align+ significantly increases the number of identified spectra as compared with MASCOT and OMSSA on both data sets. Although MS-Align+ and ProSightPC have similar performance on the Salmonella typhimurium data set, MS-Align+ outperforms ProSightPC on the (more complex) Saccharomyces cerevisiae data set.     

Human homologs of two testes-expressed loci on mouse chromosome 17 map to opposite arms of chromosome 6

Our laboratory has recently cloned and characterized two testes-expressed loci--the Tcp-10 gene family cluster and the D17Si11 gene--that map to the proximal portion of mouse chromosome 17. Human homologs of both loci have been identified and cloned. Somatic cell hybrid lines have been used to map the human homolog of D17Si11 to the short arm of chromosome 6 (p11-p21.1) along with homologs of other genes from the (Pim-1)-(Pgk-2) region of the mouse chromosome. The human TCP 10 locus maps to the long arm of chromosome 6 (q21-qter) along with homologs of other genes from the mouse chromosome 17 region between the centromere and Pim-1. The mapping of large portions of the mouse t haplotype to unlinked regions on human chromosome 6 rules out the possibility that a t-haplotype-like chromosome could exist in humans.     

Mechanism of adenylate kinase. Structural and functional demonstration of arginine-138 as a key catalytic residue that cannot be replaced by lysine

Replacement of the arginine-138 of adenylate kinase (AK) by lysine or methionine resulted in a decrease in kcat by a factor of 10(4), increases in Km by a factor of 10-20, and relatively little changes in dissociation constants. Proton nuclear magnetic resonance (NMR) studies were then undertaken to obtain structural information for quantitative interpretation of the kinetic data. Since the lysine mutant (R138K) represents a conservative mutation with surprisingly large effects on kinetics, structural studies were focused on the wild type (WT) and R138K. The results and conclusions are summarized as follows: (i) The aromatic spin systems of WT and R138K were assigned from total correlated spectroscopy (TOCSY). Comparison of the chemical shifts of aromatic protons, one-dimensional spectra, TOCSY, and nuclear Overhauser enhanced spectroscopy (NOESY) indicated that the conformation of R138K was almost unperturbed relative to that of WT. Thus Arg-138 is not important for the tertiary structure. (ii) Proton NMR titrations with AMP and MgATP suggested that substrate binding affinities and substrate-induced conformational changes are nearly identical between WT and R138K. Thus arginine-138 should not be involved in stabilizing the first substrate in the binary complex. (iii) Notable differences were observed between the proton NMR spectra of the WT and R138K complexes with the reaction mixture, which agrees with the perturbation in the Km values of R138K. The differences were analyzed in detail by using a "static reaction mixture'--p1, p5-bis(5'-adenosyl)pentaphosphate (MgAP5A). The aromatic spin systems of WT + MgAP5A and R138K + MgAP5A were partially assigned from various two-dimensional spectra.(ABSTRACT TRUNCATED AT 250 WORDS)     

2,3-Dimethoxybenzo[i]phenanthridines: topoisomerase I-targeting anticancer agents

Appropriately substituted benzo[i]phenanthridines structurally related to nitidine, a benzo[c]phenanthridine alkaloid with antitumor activity, are active as topoisomerase I-targeting agents. Studies on benzo[i]phenanthridines have indicated analogues that possess a 2,3-methylenedioxy moiety and at least one and preferably two methoxyl groups at the 8- and 9-positions, such as 8,9-dimethoxy-2,3-methylenedioxybenzo[i]phenanthridine, 2, are active as topoisomerase I-targeting agents. Tetramethoxylated benzo[i]phenanthridines, wherein the 2,3-methylenedioxy moiety is replaced with methoxyl groups at the 2- and 3-position, are inactive as a topoisomerase I-targeting agent. These results initially suggested that the 2,3-methylenedioxy moiety was critical to the retention of potent activity. Further studies revealed that 2,3-dimethoxy-8,9-methylenedioxybenzo[i]phenanthridine, 7a, is more potent than 2 as a topoisomerase I-targeting agent. The observation that 2,3-dimethoxylated benzo[i]phenanthridines can actually exhibit enhanced activity prompted the present study in which several 8-substituted 2,3-dimethoxybenzo[i]phenanthridines were prepared and their pharmacological activities evaluated. The influence of NH(2), CN, CH(2)OH, OBn, OCH(3), OH, and NHCOCH(3 )substituents at the 8-position on the relative activity of these 2,3-dimethoxybenzo[i]phenanthridines was examined. Relative to these derivatives, 7a was the most potent topoisomerase I-targeting agent, possessing similar cytotoxicity to that of nitidine in the human lymphoblast tumor cell line, RPMI8402.     

Detecting Minimal Hepatic Encephalopathy in an Endemic Country for Hepatitis B: The Role of Psychometrics and Serum IL-6

Background & Aims

It remains unknown what the prevalence of minimal hepatic encephalopathy is in Taiwan, a highly endemic country for chronic viral hepatitis infection. It is also unclear whether abnormal serum cytokine levels can be indicative of the presence of minimal hepatic encephalopathy. We aimed to standardize the tests of psychometric hepatic encephalopathy score and predictive value of proinflammatory cytokines in minimal hepatic encephalopathy in Taiwan.

Methods

180 healthy subjects and 94 cirrhotic patients without a history of overt hepatic encephalopathy from a tertiary center were invited to participate in this cross-sectional study. Blood sampling for determination of serum levels of interleukin 6 and 18 and tumor necrosis factor-α was performed. Based on the normogram of psychometric hepatic encephalopathy score from healthy volunteers, patients with minimal hepatic encephalopathy were identified from the cirrhotic patients using the criterion of a psychometric hepatic encephalopathy score less than −4.

Results

In the healthy subjects, age and education were predictors of subtests of psychometric hepatic encephalopathy score. Minimal hepatic encephalopathy was identified in 27 (29%) cirrhotic patients. Serum interleukin 6 level (OR = 6.50, 95% CI = 1.64–25.76, P = 0.008) was predictive of the presence of minimal hepatic encephalopathy after multivariate analysis.

Conclusions

The psychometric hepatic encephalopathy score can be a useful tool for detecting patients with minimal hepatic encephalopathy in Taiwan and around one third of cirrhotic outpatients fulfill this diagnosis. A high serum interleukin 6 level is predictive of the presence of minimal hepatic encephalopathy.