Recyclable magnetite nanoparticle coated with cationic polymers for adsorption of DNA.

Magnetite nanoparticle (MNP) grafted with a cationic copolymer between poly(2-(N,N-diethylamino) ethyl methacrylate) and poly(poly(ethylene glycol) methyl ether methacrylate)) for efficient and recyclable adsorption of 5'-fluorescein-tagged DNA (FAM-dT9) was prepared. MNP having highest degree of positive charge (+32.1 ± 1.9 mV) retained 100% adsorption of FAM-dT9 during eight adsorption-separation-desorption cycles. The MNP having lower degree of positive charge showed a slight decrease in adsorption percentages (94-98% adsorption) after multiple recycling processes. This biocompatible hybrid material with charged surface and magnetic-responsive properties might be applicable for use as a nanosolid support for efficient and facile separation of various bioentities.

Magnetite nanoparticle with positively charged surface for immobilization of peptide nucleic acid and deoxyribonucleic acid.

We herein report the surface modification of magnetite nanoparticle (MNP) with the (co)polymer of poly(ethylene glycol) methyl ether methacrylate (PEGMA) and/or diethylamino ethyl methacrylate (DEAEMA) via atom transfer radical polymerization (ATRP) for use as anion exchanger solid support for detection of DNA sequence using peptide nucleic acid (PNA) probe. Molar ratio of the PEGMA:DEAEMA (co)polymer was systematically varied to tune the positive charges on the particle surface. Kinetic studies of the (co)polymerizations were investigated via 1HNMR to disclose the relative reactivity of the (co)polymers in the reaction. Zeta potential of the (co)polymer-grafted MNP was analyzed by photo correlation spectroscopy (PCS). Transmission electron microscopy (TEM) and PCS indicated an improvement in the particle dispersibility in water upon quaternization of the DEAEMA entities grafted on the particle surface. From the preliminary results, these (co)polymer-grafted MNPs can be used as a nanosolid support to differentiate between full match and single-base mismatch DNA sequences using an acpcPNA probe. These novel cationic MNPs might be efficiently applicable for use as a magnetically guidable tool for detection of DNA sequences.

A nested sequence-specific primer-polymerase chain reaction for the detection of HLA-B*15:02.

In this study, we reported a new technique in detecting HLA-B*15:02 by using a nested sequence-specific primer-polymerase chain reaction (SSP-PCR) that can be used on genomic DNA and whole blood for carbamazepine hypersensitivity prediction. We tested a total of 200 blind samples with known human leukocyte antigen (HLA)-B allelic types (44 positive for HLA-B*15:02 and 156 negative for HLA-B*15:02) with this new nested SSP-PCR technique and compared its efficacy to that of commercial sequence-specific oligonucleotide probe-polymerase chain reaction (SSOP-PCR). Using starting materials from DNA and whole blood, we were able to detect HLA-B*15:02 in 44 of our samples correctly. The test is very sensitive and is highly reproducible.

Malarial (Plasmodium falciparum) dihydrofolate reductase-thymidylate synthase: structural basis for antifolate resistance and development of effective inhibitors.

Dihydrofolate reductase-thymidylate synthase (DHFR-TS) from Plasmodium falciparum, a validated target for antifolate antimalarials, is a dimeric enzyme with interdomain interactions significantly mediated by the junction region as well as the Plasmodium-specific additional sequences (inserts) in the DHFR domain. The X-ray structures of both the wild-type and mutant enzymes associated with drug resistance, in complex with either a drug which lost, or which still retains, effectiveness for the mutants, reveal features which explain the basis of drug resistance resulting from mutations around the active site. Binding of rigid inhibitors like pyrimethamine and cycloguanil to the enzyme active site is affected by steric conflict with the side-chains of mutated residues 108 and 16, as well as by changes in the main chain configuration. The role of important residues on binding of inhibitors and substrates was further elucidated by site-directed and random mutagenesis studies. Guided by the active site structure and modes of inhibitor binding, new inhibitors with high affinity against both wild-type and mutant enzymes have been designed and synthesized, some of which have very potent anti-malarial activities against drug-resistant P. falciparum bearing the mutant enzymes.

Synthesis of 2-[4'-(ethylcarbamoyl)phenyl]-N-acetylglycine, the proposed structure for giganticine.

A compound (1) with the structure proposed for giganticine, an antifeedant principle isolated from the root bark of Caloropis gigantea, has been successfully synthesized by two independent methods. Comparison of physical properties and spectroscopic data of 1 with giganticine revealed that they are different compounds. All available evidence suggests that the proposed structure of giganticine is incorrect.

Cytotoxic labdane diterpenoids from Croton oblongifolius.

Three labdane diterpenoids, 2-acetoxy-3-hydroxy-labda-8(17),12(E)-14-triene, 3-acetoxy-2-hydroxy-labda-8(17),12(E)-14-triene, and 2,3-dihydroxy-labda-8(17),12(E),14-triene were isolated from stem bark of Croton oblongifolius. Their structures were established by spectroscopic data, and each was also tested for cytotoxicity against various human tumor cell lines. The latter compound showed non-specific, moderate, cytotoxicities against all the cell lines; whereas the first two compounds were less active.

Synthesis and properties of chiral peptide nucleic acids with a N-aminoethyl-D-proline backbone.

A synthon of D-proline substituted at the 4-position by thymine and at N by a flexible aminoethyl linker, has been used to prepare a novel chiral peptide nucleic acid (cPNA) with (2R,4R) stereochemistry using solid phase methodology. The homothymine decamer cPNA binds to complementary polyadenylic acid to form a 2:1 hybrid with high affinity and specificity according to UV and CD studies, whereas no binding to the corresponding polydeoxyadenylic acid was observed.

Interaction of pyrimethamine, cycloguanil, WR99210 and their analogues with Plasmodium falciparum dihydrofolate reductase: structural basis of antifolate resistance.

The nature of the interactions between Plasmodium falciparum dihydrofolate reductase (pfDHFR) and antimalarial antifolates, i.e., pyrimethamine (Pyr), cycloguanil (Cyc) and WR99210 including some of their analogues, was investigated by molecular modeling in conjunction with the determination of the inhibition constants (Ki). A three-dimensional structural model of pfDHFR was constructed using multiple sequence alignment and homology modeling procedures, followed by extensive molecular dynamics calculations. Mutations at amino acid residues 16 and 108 known to be associated with antifolate resistance were introduced into the structure, and the interactions of the inhibitors with the enzymes were assessed by docking and molecular dynamics for both wild-type and mutant DHFRs. The Ki values of a number of analogues tested support the validity of the model. A 'steric constraint' hypothesis is proposed to explain the structural basis of the antifolate resistance.

Development of a lead inhibitor for the A16V+S108T mutant of dihydrofolate reductase from the cycloguanil-resistant strain (T9/94) of Plasmodium falciparum.

The Ala16Val+Ser108Thr (A16V+S108T) mutant of the Plasmodium falciparum dihydrofolate reductase (DHFR) is a key mutant responsible for cycloguanil-resistant malaria due to steric interaction between Val-16 and one of the C-2 methyl groups of cycloguanil. 4,6-Diamino-1,2-dihydrotriazines have been prepared, in which both methyl groups of cycloguanil are replaced by H or by H and an alkyl or phenyl group, and their inhibition constants against wild-type and mutant DHFR determined. The S108T mutation is considered to decrease cycloguanil binding further through the effect on the orientation of the p-chlorophenyl group. By moving the p-chloro-substituent to the m-position in the chlorophenyl group, the activity against the A16V+S108T mutant enzyme is improved, and this effect is reinforced by the p-chloro substituent in the 3, 4-dichlorophenyl group. A lead compound has been found with inhibitory activity similar to that of cycloguanil against the wild-type DHFR and about 120-fold more effective than cycloguanil against the A16V+S108T mutant enzyme. The activity of this compound against P. falciparum clone (T9/94 RC17) which harbors the A16V+S108T DHFR is about 85-fold greater than cycloguanil.

Immunochemical analysis and immunocytochemical localization of crustacean hyperglycemic hormone from the eyestalk of Macrobrachium rosenbergii.

Heptapeptide (YANAVQV-NH2 = T-) and octapeptide (YANAVQTV-NH2 = T+), the putative C-terminus of crustacean hyperglycemic hormone (CHH) from the eyestalk of the giant freshwater prawn Macrobrachium rosenbergii, was synthesized by solid phase peptide synthesis and conjugated to bovine serum albumin, then used for immunization in swiss mice. Specificity of the antisera against both peptides was determined by indirect immunoperoxidase ELISA. The best response of antiserum against each peptide was used to determine the presence of the natural CHH in the eyestalk extract after separation by one step of RP-HPLC using dot-ELISA. The peptide immunoreactive substances were found in fraction 30 using anti-T- antiserum and in fraction 38 using anti-T+ antiserum. However, the CHH activity was found only in fractions 37-39. Immunocytochemical localization of peptide immunoreactive substances in the eyestalk of M. rosenbergii using the anti-T- antiserum did not show any specific staining. In contrast, the anti-T+ antiserum revealed specific staining on a group of 24 +/- 5 neurons in medulla terminalis ganglionic x-organ and their processes through the sinus gland. Similar results were also obtained using the eyestalk of another species, the giant tiger prawn Penaeus monodon, in which 34 +/- 4 neuronal cells were recognized. These results strongly indicate that the anti-T+ antibody can bind to the natural CHH while the anti-T- antibody can not; therefore, this isoform of CHH in M. rosenbergii should consist of 72 residues and threonine is predicted to be present at position 71.

Cytotoxicity of 2,2':6',2' '-terpyridineplatinum(II) complexes against human ovarian carcinoma.

2,2':6',2''-Terpyridineplatinum(II) complexes are shown to possess cytotoxicity against a number of human ovarian tumor cell lines. Many of the complexes show similar activity against cisplatin- and doxorubicin-resistant cell lines as the parental cells suggesting that there is little or no cross-resistance with cisplatin or doxorubicin. The cytotoxicity of bis[2,2':6',2''-terpyridineplatinum(II)] complexes is strongly dependent on the nature of the linker. Bis[2,2':6',2''-terpyridineplatinum(II)] complexes with a flexible linker at the 4'-position show poor cytotoxicity; by contrast bis[2,2':6',2''- terpyridineplatinum(II)] complexes with rigid and short linkers at platinum(II) are strikingly effective. Several of the compounds show greater cytotoxicity against human ovarian cell lines than carboplatin, the therapeutic agent currently advocated for the treatment of human ovarian cancers.

Cytotoxicity of (2,2':6',2''-terpyridine)platinum(II) complexes to Leishmania donovani, Trypanosoma cruzi, and Trypanosoma brucei.

A range of (2,2':6',2''-terpyridine)platinum(II) complexes are shown to possess antiprotozoal activity in vitro against Leishmania donovani, Trypanosoma cruzi, and Trypanosoma brucei,the causative organisms of tropical diseases leishmaniasis and trypanosomiasis. The best compounds caused 100% and 78% inhibition of growth of the intracellular amastigote forms of L. donovani and T. cruzi, respectively, at a concentration of 1 microM and 100% inhibition of growth of the bloodstream trypomastigote forms of T. brucei at a concentration of 0.03 microM. The results obtained with complexes in which the fourth ligand to platinum(II) is capable of being substituted with a substitution inert hydroxyethanethiolate complex are compared. The ammine complexes show high antiprotozoal activity suggesting that the trans influence of the 2,2':6',2''-terpyridine ligand has a profound effect on the ease of displacement of the fourth ligand in (2,2':6',2'' -terpyridine)platinum(II) complexes, although nonbonded interaction between the ammine ligand and the 6 and 6' ' hydrogens probably also weakens the ligation to Pt(II).

Two new cembranoids from croton oblongifolius

Two new cembranoids, crotocembraneic acid (1) and neocrotocembraneic acid (2), were isolated from the stem bark of Croton oblongifolius. Their structures were established on the basis of spectroscopic analysis.

A mass spectrometric investigation of the reaction between 4,4'-vinylenedipyridine bis[2,2':6',2"-terpyridine platinum(II)] and the self-complementary oligonucleotide d(CpGpTpApCpG).

4,4'-Vinylenedipyridine bis[2,2':6',2"-terpyridine platinum(II)], a potential intermolecular bis-intercalator of DNA, reacts slowly at PtII, the linker being displaced by nucleobases. Crystallization of 4,4'-vinylenedipyridine bis[2,2':6',2"-terpyridine platinum(II)] in the presence of the self-complementary oligonucleotide d(CpGpTpApCpG) gave a product which was analyzed by electrospray ionization mass spectrometry. Molecular mass measurements demonstrated ligation of one terpyridine PtII, which was shown by liquid chromatography-mass spectrometry to reside exclusively on deoxyguanosine. The terpyridine PtII was shown by tandem mass spectrometry to be attached to the 3'-terminal deoxyguanosine, consistent with N-7 substitution.