Compounds of Mo, V and W in biochemistry and their biomedical activity.

Molybdenum, vanadium and tungsten compounds are widely applied as analytical reagents for determination of numerous pharmacologically active substances and different biochemical parameters. Recent data from the available literature pointed to a very potent biomedical activity of compounds containing these trace elements. The present paper represents a survey on the structure and chemical properties of these compounds, as well as on their biological activity, mostly based on their interaction with cations of biomolecules, such as phospholipids and proteins. Besides, their potent inhibitory effects on cellular targets, bacterial and viral DNA and RNA polymerases will be discussed, as well. Numerous authors clearly demonstrated the antiviral (especially anti-HIV), anticoagulant and antineoplastic properties of the compounds containing the above trace elements. It has been also shown that these compounds act on some cellular enzymatic systems leading to the normalisation of blood pressure, blood glucose and serum lipid levels. Also, compounds of these trace elements represent potent antiobesity agents and express hepatoprotective and antioxidative stress activity.     

DNA-dependent RNA polymerases isolated from yeast mitochondria

Purified preparations of yeast mitochondria yield three species of DNA-dependent RNA polymerases. These enzymes have been separated and purified to homogeneity for analysis of their properties and for comparison with the properties of nuclear preparations of yeast RNA polymerases. Three enzymes have been separated by DEAE-Sephadex chromatography of each fraction. Both nuclear and mitochondrial preparations yield three components with nearly identical elution properties. The distributions of enzyme activity on DEAE-Sephadex chromatography differ with the three nuclear peaks, being found in ratios (uncorrected for the effect of increasing salt concentration) of 8:85:7 and the mitochondrial peaks in ratios of 8:32:60 at late log phase of growth under optimized conditions in which protease inhibitors and an antioxidant were included. The type of mitochondrial enzymes in 3-day-old cells differed from those grown to late logarithmic phase. It has been established that the enzymes of the mitochondrial preparation are associated with the membrane fraction. While extraction with 0.5 m KCl solubilizes considerable enzyme activity, greatly enhanced yields of enzyme MIII are obtained by addition of the antioxidant 2,6-di-t-butyl-4-hydroxymethyl phenol during enzyme extraction. Inhibition of protease activity has also been shown to have a major effect on the yield and distribution of enzymes obtained from mitochondrial preparations. The mitochondrial preparations of yeast polymerases are generally similar but not identical to corresponding nuclear polymerases in subunit molecular weights, inhibitor sensitivities, and in DNA template dependence. Comparative studies of nuclear and mitochondrial polymerases clearly establish that differences do exist among the isolated enzymes of these classes. It has not been ruled out to date that these enzymes may be derived in part or in total from the same cytoplasmic subunit pool, nor has it been established that any of these enzymes function in mitochondria in vivo.     

Eukaryotic DNA polymerases

Knowledge about eukaryotic DNA polymerases has increased considerably during recent years. Much have been learnt about both the structures and the functions of "classical" DNA polymerases alpha, beta, delta, epsilon and gamma. New DNA polymerases that possess very unusual functions have been identified. They are able to perform translesional synthesis, take part in somatic hypermutation and prevent some cancers. Much attention has also been devoted to the role of 3'-->5' exonuclease activity in the accuracy of DNA synthesis. On the other hand, it have been shown that there are also negative aspects of the activity of DNA polymerases. Lack of some DNA polymerases or even their altered functions may lead to carcinogenesis and accelerate the process of ageing.     

Quantitative conservation of chromatin-bound RNA polymerases I and II in mitosis. Implications for chromosome structure

RNA synthesis almost ceases in mitosis. It is ambiguous whether this temporal, negative control of RNA synthesis is solely because of the nature of chromosomes per se, (i.e., their condensed state), or to a physical loss of RNA polymerases along with other nuclear proteins which have been shown to pass into the cytoplasm in mitosis, or to their combined feature. Aside from such regulatory considerations, a question has also been raised as to whether RNA polymerases are constituents of metaphase chromosomes. To clarify these aspects of RNA polymerase-chromatin interaction in mitosis, the enzymes in chromosomes were quantitated and their levels compared to those in interphase nuclei and cells at various phases of the cell cycle. The results show that the amounts of form I, form II, and probably form III enzymes bound to a genome-equivalent of chromatin stay constant during the cell cycle. Thus, the mechanism for the negative control of RNA synthesis in mitosis appears to exist in the chromosomes per se, but not to be directly related to the RNA polymerase levels. This quantitative conservation of chromatin-bound RNA polymerases implies that they may persist as structural components of the chromosomes in mitosis.     

THE ACCUMULATION OF ELECTROLYTES : VII. ORGANIC ELECTROLYTES PART 2

Analyses have been made of the inorganic constituents of the juices expressed from the leaves of Rheum, Rumex, and Oxalis. It has been shown that in all cases there is a large excess of inorganic cations over anions in the sap, the average ratio of cations to anions being 3.8 (Part 1, p. 239). The ash analyses of plant tissues (chiefly leaves) reported in the literature have been examined critically, and it has been shown that the preponderance of inorganic cations over inorganic anions in the ash and in the sap is general. It has been concluded that the excess of inorganic cations is consistent with the view that cations pass into the protoplasm chiefly in the form of hydroxides, and are accumulated either in the form of organic salts (such as the oxalates) or in non-polar linkage. It has been concluded that practically all the potassium and sodium found in plant ash must have been present originally in the form of soluble ionogenic compounds, but that a considerable part of the calcium and magnesium may have been present originally in the form of insoluble salts or as components of non-polar compounds. The methods whereby the cations, particularly potassium, may have been accumulated have been discussed, and it has been concluded that as it does not seem very probable that they enter chiefly as nitrates or bicarbonates we may suppose that they go in to a large extent as hydrates: this is highly probable in the case which has been most carefully investigated (Valonia).     

Action of strong and weak nitrogenous bases on respiratory chain enzymes of mitochondria

The action of nitrogenous basis--electroneutral hydrazides (pK less than 7,50 and positive charged arylhydrazones (pK greater than 8)--on the respiratory chain enzymes and the influence of the electric charge and the size of alkoxylic group on biological activity compounds have been investigated. It has been shown that the size of alkoxylic group defines the selective action of nitrogenous basis on the enzymes of mitochondrial respiratory chain. The nitrogenous basis with a long alkoxylic group is shown to be inhibitors of NADH-dehydrogenase, their action is similar to rotenone. At the same time compounds with a short group are more effective in the inhibition of the enzymes of the initial segment in the respiratory chain mitochondria. The affinity of the organic cations of arylhydrazones to NADH-dehydrogenase is 100-1000 times higher than the affinity of electric neutral compounds.     

Physical mapping of drug resistance mutations defines an active center of the herpes simplex virus DNA polymerase enzyme.

The genome structures of herpes simplex virus type 1 (HSV-1)/HSV-2 intertypic recombinants have been previously determined by restriction endonuclease analysis, and these recombinants and their parental strains have been employed to demonstrate that mutations within the HSV DNA polymerase locus induce an altered HSV DNA polymerase activity, exhibiting resistance to three inhibitors of DNA polymerase. The viral DNA polymerases induced by two recombinants and their parental strains were purified and shown to possess similar molecular weights (142,000 to 144,000) and similar sensitivity to compounds which distinguish viral and cellular DNA polymerases. The HSV DNA polymerases induced by the resistant recombinant and the resistant parental strain were resistant to inhibition by phosphonoacetic acid, acycloguanosine triphosphate, and the 2',3'-dideoxynucleoside triphosphates. The resistant recombinant (R6-34) induced as much acycloguanosine triphosphate as did the sensitive recombinant (R6-26), but viral DNA synthesis in infected cells and the viral DNA polymerase activity were not inhibited. The 2',3'-dideoxynucleoside-triphosphates were effective competitive inhibitors for the HSV DNA polymerase, and the Ki values for the four 2',3'-dideoxynucleoside triphosphates were determined for the four viral DNA polymerases. The polymerases of the resistant recombinant and the resistant parent possessed a much higher Ki for the 2',3'-dideoxynucleoside triphosphates and for phosphonoacetic acid than did the sensitive strains. A 1.3-kilobase-pair region of HSV-1 DNA within the HSV DNA polymerase locus contained mutations which conferred resistance to three DNA polymerase inhibitors. This region of DNA sequences encoded for an amino acid sequence of 42,000 molecular weight and defined an active center of the HSV DNA polymerase enzyme.     

Synthesis and biological evaluation of aromatic enones related to curcumin

Curcumin, a natural product isolated from the spice turmeric, has been shown to exhibit a wide range of pharmacological activities including certain anti-cancer properties. It has been specifically shown to be an effective inhibitor of angiogenesis both in vitro and in vivo. Using curcumin as a lead compound for anti-angiogenic analog design, a series of structurally related compounds utilizing a substituted chalcone backbone have been synthesized and tested via an established SVR cell proliferation assay. The results have yielded a wide range of compounds that equal or exceed curcumin's ability to inhibit endothelial cell growth in vitro. Due to both their commercial availability and their fairly straightforward synthetic preparation, these low molecular weight compounds are attractive leads for developing future angiogenic inhibitors.     

Structural and functional relationships of human DNA polymerases

A continuing theme of our laboraory, has been the understanding of human DNA polymerases at the structural level. We have purified DNA polymerases delta, epsilon and alpha from human placenta. Monoclonal antibodies to these polymerases were isolated and used as tools to study their immunochemical relationships. These studies have shown that while DNA polymerases delta, epsilon and alpha are discrete protiens, they must share common structural features by virtue of the ability of several of our monoclonal antibodies to exhibit cross-reactivity. A second approach we have taken is the molecular cloning of human DNA polymerase delta and epsilon. We have cloned the DNA polymerase delta cDNA, and this has allowed us to compare its primary structure to those of human polymerase alpha and other members of this polymerase family. Multiple sequence alignments have revealed that human DNA polymerase delta is also closely related to the herpes virus family of DNA polymerases. In situ hybridization has shown that the human DNA polymerase delta gene is localized to chromosome 19 q13.3–q13.4. In order to further determine the functional regions of the DNA polymerase δ structure we are currently expressing human pol δ inE. coli and baculovirus systems. Other work in our laboratory is directed toward examining the expression of DNA polymerase δ during the cell cycle.     

跨损伤合成的DNA聚合酶——一类新的DNA聚合酶

细胞虽然拥有多种修复途径,但有些DNA损伤仍不可避免地会逃避修复而在基因组上保留下来,细胞跨损伤DNA合成的分子机制一直是DNA修复中主要的未解决问题之一.最近通过对一类结构相关性UmuC/DinB蛋白质超家族成员的研究发现它们具有DNA聚合酶功能.这类新发现的DNA聚合酶不同于经典的复制性DNA聚合酶,它们能以易误/突变(error-prone/mutagenic)或无误(error-free)方式进行跨损伤(translesion)DNA合成,并且从细菌到人在进化上功能保守.     

Structural and functional relationships of human DNA polymerases

A continuing theme of our laboraory, has been the understanding of human DNA polymerases at the structural level. We have purified DNA polymerases delta, epsilon and alpha from human placenta. Monoclonal antibodies to these polymerases were isolated and used as tools to study their immunochemical relationships. These studies have shown that while DNA polymerases delta, epsilon and alpha are discrete protiens, they must share common structural features by virtue of the ability of several of our monoclonal antibodies to exhibit cross-reactivity. A second approach we have taken is the molecular cloning of human DNA polymerase delta and epsilon. We have cloned the DNA polymerase delta cDNA, and this has allowed us to compare its primary structure to those of human polymerase alpha and other members of this polymerase family. Multiple sequence alignments have revealed that human DNA polymerase delta is also closely related to the herpes virus family of DNA polymerases. In situ hybridization has shown that the human DNA polymerase delta gene is localized to chromosome 19 q13.3–q13.4. In order to further determine the functional regions of the DNA polymerase δ structure we are currently expressing human pol δ inE. coli and baculovirus systems. Other work in our laboratory is directed toward examining the expression of DNA polymerase δ during the cell cycle.     

Directed polymerase evolution

Polymerases evolved in nature to synthesize DNA and RNA, and they underlie the storage and flow of genetic information in all cells. The availability of these enzymes for use at the bench has driven a revolution in biotechnology and medicinal research; however, polymerases did not evolve to function efficiently under the conditions required for some applications and their high substrate fidelity precludes their use for most applications that involve modified substrates. To circumvent these limitations, researchers have turned to directed evolution to tailor the properties and/or substrate repertoire of polymerases for different applications, and several systems have been developed for this purpose. These systems draw on different methods of creating a pool of randomly mutated polymerases and are differentiated by the process used to isolate the most fit members. A variety of polymerases have been evolved, providing new or improved functionality, as well as interesting new insight into the factors governing activity.     

DNA lesion bypass polymerases and 4’-thio-β-Darabinofuranosylcytosine (T-araC)

The 4’-thio-β-D-arabinofuranosylcytosine (T-araC) is a newly developed nucleoside analog that has shown promising activity against a broad spectrum of human solid tumors in both cellular and xenograft mice models. TaraC shares similar structure with another anticancer deoxycytidine analog, β-D-arabinofuranosylcytosine (araC, cytarabine), which has been used in clinics for the treatment of acute myelogenous leukemia but has a very limited efficacy against solid tumors. T-araC exerts its anticancer activity mainly by inhibiting replicative DNA polymerases from further extension after its incorporation into DNA. DNA lesion bypass polymerases can manage the DNA lesions introduced by therapeutic agents, such as cisplatin and araC, therefore reduce the activity of these compounds. In this study, the potential relationships between the lesion bypass Y-family DNA polymerases η, ι and κ (pol η, pol ι, and pol κ) and T-araC were examined. Biochemical studies indicated that the triphosphate metabolite of T-araC is a less preferred substrate for the Y-family polymerases. In addition, cell viability study indicated that pol η deficient human fibroblast cells were more sensitive to T-araC when compared with the normal human fibroblast cells. Together, these results suggest that bypass polymerases reduced cell sensitivity to T-araC through helping cells to overcome the DNA damages introduced by T-araC.     

Heat-induced alterations in DNA polymerase activity of HeLa cells and of isolated nuclei. Relation to cell survival

The activity of DNA polymerase alpha and beta was assayed in heated HeLa S3 cells as well as in nuclei isolated from these cells. The enzyme activity as measured in cells and in nuclei has been compared with the extent of cell survival after the different hyperthermic doses. It was found that although the activity of the cellular DNA polymerases was related to cell survival after single heat doses, no correlation was found when thermotolerant cells were heated. When the activity of the DNA polymerases was determined in nuclei isolated from non-heated and heated cells, more polymerase activity was found in the nuclei of the heated cells. However, the heat sensitivity of DNA polymerase activity was the same for nuclei isolated from control, pre-heated and thermotolerant cells. Heat protection of polymerase activity by erythritol and sensitization by procaine was found when cells, but not when nuclei, were heated in the presence of these modifiers. It is concluded that (the nuclear bound) DNA polymerases are not to be considered as key enzymes in cellular heat sensitivity of HeLa S3 cells.     

Characterization of a DNA polymerase from the uncultivated psychrophilic archaeon Cenarchaeum symbiosum.

Cenarchaeum symbiosum, an archaeon which lives in specific association with a marine sponge, belongs to a recently recognized nonthermophilic crenarchaeotal group that inhabits diverse cold and temperate environments. Nonthermophilic crenarchaeotes have not yet been obtained in laboratory culture, and so their phenotypic characteristics have been inferred solely from their ecological distribution. Here we report on the first protein to be characterized from one of these organisms. The DNA polymerase gene of C. symbiosum was identified in the vicinity of the rRNA operon on a large genomic contig. Its deduced amino acid sequence is highly similar to those of the archaeal family B (alpha-type) DNA polymerases. It shared highest overall sequence similarity with the crenarchaeal DNA polymerases from the extreme thermophiles Sulfolobus acidocaldarius and Pyrodictium occultum (54% and 53%, respectively). The conserved motifs of B (alpha-)-type DNA polymerases and 3'-5' exonuclease were identified in the 845-amino-acid sequence. The 96-kDa protein was expressed in Escherichia coli and purified with affinity tags. It exhibited its highest specific activity with gapped-duplex (activated) DNA as the substrate. Single-strand- and double-strand-dependent 3'-5' exonuclease activity was detected, as was a marginal 5'-3' exonuclease activity. The enzyme was rapidly inactivated at temperatures higher than 40 degrees C, with a half-life of 10 min at 46 degrees C. It was found to be less thermostable than polymerase I of E. coli and is substantially more heat labile than its most closely related homologs from thermophilic and hyperthermophilic crenarchaeotes. Although phylogenetic studies suggest a thermophilic ancestry for C. symbiosum and its relatives, our biochemical analysis of the DNA polymerase is consistent with the postulated nonthermophilic phenotype of these crenarchaeotes, to date inferred solely from their ecological distribution.     

Inhibition of NAD+-dependent 15-hydroxyprostaglandin dehydrogenase (15-PGDH) by cyclooxygenase inhibitors and chemopreventive agents

15-Hydroxyprostaglandin dehydrogenase (15-PGDH) catalyzes NAD(+)-dependent oxidation of 15(S)-hydroxyl group of prostaglandins and has been considered a key enzyme involved in biological inactivation of prostaglandins. This enzyme is markedly induced by androgens in hormone-sensitive human prostate cancer cells (Tong M., Tai H. H. Biochem Biophys Res Commun 2000; 276: 77-81) and may be involved in tumorigenesis. Inhibition of this enzyme may be of value in anticancer therapy. Non-steroidal anti-inflammatory drugs (NSAIDs) which inhibit cyclooxygenases (COXs) have been shown to be chemopreventive in epidemiological and animal-model studies. However, chemoprevention by these drugs may not be directly related to their inhibition of COXs. Other targets may be also involved in their chemopreventive activity. We have examined a variety of NSAIDs including COX-2 selective inhibitors, peroxisome proliferator-activated receptor (PPAR) gamma agonists and phytophenolic compounds which have been shown to be chemopreventive for their effect on 15-PGDH. It was found that most of these compounds were potent inhibitors of 15-PGDH. Among these compounds, ciglitazone appeared to be the most powerful inhibitor (IC(50)=2.7 microM). Inhibition by ciglitazone was non-competitive with respect to NAD(+) and uncompetitive with respect to PGE(2).     

Isoprostanes: novel bioactive products of lipid peroxidation

Isoprostanes, are a novel group of prostaglandin-like compounds that are biosynthesised from esterified polyunsaturated fatty acid (PUFA) through a non-enzymatic free radical-catalysed reaction. Several of these compounds possess potent biological activity, as evidenced mainly through their pulmonary and renal vasoconstrictive effects, and have short half-lives. It has been shown that isoprostanes act as full or partial agonists through thromboxane receptors. Both human and experimental studies have indicated associations of isoprostanes and severe inflammatory conditions, ischemia-reperfusion, diabetes and atherosclerosis. Reports have shown that F₂-isoprostanes are authentic biomarkers of lipid peroxidation and can be used as potential in vivo indicators of oxidant stress in various clinical conditions, as well as in evaluations of antioxidants or drugs for their free radical-scavenging properties.

Higher levels of F₂-isoprostanes have been found in the normal human pregnancy compared to non-pregnancy, but their physiological role has not been well studied so far. Since bioactive F₂-isoprostanes are continuously formed in various tissues and large amounts of these potent compounds are found unmetabolised in their free acid form in the urine in normal basal conditions with a wide inter-individual variation, their role in the regulation of normal physiological functions could be of further biological interest, but has yet to be disclosed. Their potent biological activity has attracted great attention among scientists, since these compounds are found in humans and animals in both physiological and pathological conditions and can be used as reliable biomarkers of lipid peroxidation.     

The role of dicarbonyl compounds in non-enzymatic crosslinking: a structure-activity study

The Maillard reaction is a complex network of reactions that has been shown to result in the non-enzymatic crosslinking of proteins. Recent attention has focussed on the role of alpha-dicarbonyl compounds as important in vivo contributors to protein crosslinking but, despite extensive research, the molecular mechanisms of the crosslinking reaction remain open to conjecture. In particular, no relationship between the structure of the carbonyl-containing compounds and their activity as crosslinking agents has been established. In an effort to elucidate a structure-reactivity relationship, a wide range of dicarbonyl compounds, including linear, cyclic, di-aldehyde and di-ketone compounds, were reacted with the model protein ribonuclease A and their crosslinking activity assessed. Methylglyoxal and glutaraldehyde were found to be the most efficient crosslinkers, whilst closely related molecules effected crosslinking at a much lower rate. Cyclopentan-1,2-dione was also shown to be a reactive crosslinking agent. The efficiency of methylglyoxal and glutaraldehyde at crosslinking is thought to be related to their ability to form stable heterocyclic compounds that are the basis of protein crosslinks. The reasons for the striking reactivity of these two compounds, compared to closely related structures is explained by subtle balances between competing pathways in a complex reaction network.