A novel 10B-enriched carboranyl-containing phthalocyanine as a radio- and photo-sensitising agent for boron neutron capture therapy and photodynamic therapy of tumours: in vitro and in vivo studies.

The synthesis of a Zn(ii)-phthalocyanine derivative bearing four 10B-enriched o-carboranyl units (10B-ZnB4Pc) and its natural isotopic abundance analogue (ZnB4Pc) in the peripheral positions of the tetraazaisoindole macrocycle is presented. The photophysical properties of ZnB4Pc, as tested against model biological systems, were found to be similar with those typical of other photodynamically active porphyrin-type photosensitisers, including a singlet oxygen quantum yield of 0.67. The carboranyl-carrying phthalocyanine was efficiently accumulated by B16F1 melanotic melanoma cells in vitro, appeared to be partitioned in at least some subcellular organelles and, upon red light irradiation, induced extensive cell mortality. Moreover, ZnB4Pc, once i.v.-injected to C57BL/6 mice bearing a subcutaneously transplanted pigmented melanoma, photosensitised an important tumour response, provided that the irradiation at 600-700 nm was performed 3 h after the phthalocyanine administration, when appreciable concentrations of ZnB4Pc were still present in the serum. Analogously, irradiation of the 10B-ZnB4Pc-loaded pigmented melanoma with thermal neutrons 24 h after injection led to a 4 day delay in tumour growth as compared with control untreated mice. These results open the possibility to use one chemical compound as both a photosensitising and a radiosensitising agent for the treatment of tumours by the combined application of photodynamic therapy and boron neutron capture therapy.     

VHY, a novel myristoylated testis-restricted dual specificity protein phosphatase related to VHX

The human DUSP15 gene encodes an uncharacterized 235-amino acid member of the subfamily of small dual specificity protein phosphatases related to the Vaccinia virus VH1 phosphatase. Similar to VHR-related MKPX (VHX) (DUSP22), the predicted protein has an N-terminal myristoylation recognition sequence, and we show here that both are indeed modified by the attachment of a myristate to Gly-2. In recognition of this relatedness to VHX, we refer to the DUSP15-encoded protein as VH1-related member Y (VHY). We report that VHY is expressed at high levels in the testis and barely detectable levels in the brain, spinal cord, and thyroid. A VHY-specific antiserum detected a protein with an apparent molecular mass of 26 kDa, and histochemical analysis showed that VHY was readily detectable in pachytene spermatocytes (midstage of meiotic division I) and round spermatids and weakly in Leydig cells (somatic cells outside of the seminiferous tubules). When expressed in 293T or NIH-3T3 cells, VHY was concentrated at the plasma membrane with some staining of vesicular structures in the Golgi region. Mutation of the myristoylation site Gly-2 abrogated membrane location. Finally, we demonstrate that VHY is an active phosphatase in vitro. We conclude that VHY is a new member of a subgroup of myristoylated VH1-like small dual specificity phosphatases.     

Growth and morphogenesis of embryonic mouse organs on non-coated and extracellular matrix-coated Biopore membrane

Embryonic mouse salivary glands, pancreata, and kidneys were isolated from embryos of appropriate gestational age by microdissection, and were cultured on Biopore membrane either non-coated or coated with type I collagen or Matrigel. As expected, use of Biopore membrane allowed high quality photomicroscopy of the living organs. In all organs extensive mesenchymal spreading was observed in the presence of type I collagen or Matrigel. However, differences were noted in the effects of extracellular matrix (ECM) coatings on epithelial growth and morphogenesis: salivary glands were minimally affected, pancreas morphogenesis was adversely affected, and kidney growth and branching apparently was enhanced. It is suggested that these differences in behaviour reflect differences in the strength of interactions between the mesenchymal cells and their surrounding endogenous matrix, compared to the exogenous ECM macromolecules. This method will be useful for culture of these and other embryonic organs. In particular, culture of kidney rudiments on ECM-coated Biopore offers a great improvement over previously used methods which do not allow morphogenesis to be followed in vitro.     

Characterization of foldback sequences in hamster DNA using electron microsocpy.

Foldback sequences in nuclear DNA from cultured Hamster fibroblasts (BHK-21/C13 cells) have been characterized by electron microscopy. One half of the structures observed when denatured hamster DNA is allowed to anneal in the range O less than Cot1 less than 1 x 10(-4) M sec result from the annealing of inverted sequences forming foldback DNA. The remainder have a probable bimolecular origin. arising from rapidly-annealing sequences of satellite-like complexity. The average length of the inverted sequences in the foldback molecules is about 0.9 kilobases. There is estimated to be about 42,000 such sequences (21,000 pairs) in the hamster genome, approximately 45% of which form looped structures with a mean loop length of 1.74 kilobases. Contrary to previous reports, binding of the renatured duplex molecules to hydroxyapatite results in a poor recovery of structures containing identifiable foldback sequences, due to preferential enrichment of the bound fraction with duplexes formed by intermolecular annealing.     

Sequence organisation in nuclear DNA from Physarum polycephalum: methylation of repetitive sequences.

Nuclear DNA from the slime mould Physarum polycephalum is digested by the restriction endonuclease HpaII to generate a high molecular weight and a low molecular weight component. These are referred to as the M+ and the M- compartment, respectively. Sequences that are present in the M+ compartment are cleaved by MspI, the restriction enzyme isoschizomer of HpaII, thus showing that the recognition sequences for these enzymes in M+ DNA contain methylated CpG doublets. The distribution of repetitive sequences in the M+ and M- DNA compartments was investigated by comparison of the 'fingerprint' patterns of total Physarum DNA and isolated M+ DNA after digestion using different restriction endonucleases, and by probing for the presence of specific repetitive sequences in Southern blots of M+ and M- DNA by the use of cloned DNA segments. Both types of experiment indicate that many repetitive sequences are shared by both compartments, though some repetitive sequences appear to be considerably enriched, or are present exclusively, either in M+ DNA or in M- DNA.     

Chemical, photochemical and spectroscopic characterization of an alkaline proteinase from Bacillus subtilis variant DY

Circular-dichroism and fluorescence studies indicate that the 5-dimethylaminonaphthalene-1-sulphonyl and phenylmethanesulphonyl derivatives of subtilisin DY have three-dimensional structure closely similar to that of native enzyme. The single tryptophan residue is largely accessible to the aqueous solvent, and is not directly involved in the enzyme-substrate interactions, since its photochemical modification causes only a partial inhibition of the enzyme activity. It appears very likely that the location of the single tryptophan residue in the three-dimensional structure of subtilisin DY is similar to that of the single tryptophan residue in subtilisin Carlsberg. Fluorescence-quenching experiments further indicate that the 14 tyrosine residues are also largely accessible to the aqueous solvent, and probably interact with hydrated peptide carbonyl groups. The charge environment for tryptophan and tyrosine residues in subtilisin DY, as deduced by quenching experiments with ionic species, is also discussed. In general, subtilisin DY displays strong similarities to subtilisin Carlsberg, as suggested by a comparative analysis of the amino acid composition and fluorescence properties.     

Effects of pH and urea on the conformational properties of subtilisin DY

Subtilisin DY is very resistant to the denaturing action of urea: the conformational properties are not affected up to 4.5 M-urea, and even in the presence of 8 M-urea there is only a slow loss of ordered structure and caseinolytic activity. C.d. and fluorescence-emission studies also show that this proteinase is stable in the 5.5-10.0 pH range, whereas below pH 5.5 a sharp denaturation occurs that is complete at pH 4.5. Protein denaturation leads to a change of the emission quantum yield; in particular, in the native protein, indole fluorescence is quenched by some amino groups. Moreover, subtilisin DY possesses two classes of tyrosine residues: one class of exposed residues titrates normally, with pKapp. = 10.24, whereas one class of partially buried or hydrogen-bonded residues ionizes with pKapp. = 11.58. In general, such conformational properties resemble those of other subtilisins. However, some differences occur: e.g., subtilisin DY is less stable at acidic pH values and its tyrosine residues are more accessible to the solvent. Such differences are probably due to small variations of the three-dimensional structure; e.g., subtilisin DY has a slightly lower alpha-helix content.     

The effect of chromatin structure on cisplatin damage in intact human cells

The influence of chromatin structure on cisplatin DNA damage was investigated in intact human cells. The epsilon-globin gene promoter was utilised as the target DNA sequence and the terminal transferase-dependent PCR technique was employed to examine adduct formation at base pair resolution. It was found that cisplatin preferentially damaged at runs of consecutive guanine bases in intact cells. By comparing the relative intensity of adduct formation in intact cells and in purified genomic DNA, it was possible to assess the influence of chromatin proteins on the extent of cisplatin DNA damage. Enhanced damage in intact cells was found at the CACC site where a member of the Sp1 family of proteins is thought to bind. It is postulated that protein binding at this site bends the DNA double-helix so that enhanced cisplatin binding occurs. The altered DNA binding of cisplatin in the presence of chromatin proteins could be important in the properties of cisplatin as an anti-tumour drug.     

Performance of a pyrethroid-resistant strain of the predator mite Typhlodromus pyri (Acari: Phytoseiidae) under different insecticide regimes

An organophosphate pyrethroid-resistant strain of Typhlodromus pyri Scheuten imported from New Zealand was reared on potted apple trees in an outdoor insectary. From 1988 to 1995, the population was selected one to three times per year with a dilute solution (1.7 ppm) of the pyrethroid cypermethrin. Petri dish bioassays with cypermethrin in 1995 indicated that the insectary-reared T. pyri had an LC50 of 81 ppm versus 0.006 ppm for native T. pyri taken from a research orchard. The bioassays suggested that recommended orchard rates of cypermethrin would cause heavy mortality in native populations of T. pyri but only moderate losses in the imported New Zealand strain. Bioassays in 1996 with the organophosphate insecticide dimethoate indicated both New Zealand and native T. pyri were susceptible and that recommended orchard rates of dimethoate likely would cause high mortality of T. pyri in apple orchards. These findings from bioassays were supported by data from orchard trials. In June and July 1993, insectary-reared New Zealand T. pyri were placed on five apple trees in each of eight 38-tree plots in the research orchard. In late August 1994, New Zealand T. pyri from orchard trees that had been sprayed twice by airblast sprayer with the full recommended rate of 50 g (AI)/ha (83 ppm) cypermethrin were placed on the other 33 trees in each of six plots. In the summers of 1994-1996, plots were treated with one of the following insecticide regimes: (1) conventional integrated pest management (IPM) (registered neurotoxic insecticides considered harmless or slightly toxic to T. pyri); (2) advanced IPM (use of newer, more selective insecticides); (3) pyrethroid (at least one full-rate application of cypermethrin); (4) dimethoate; and (5) dimethoate plus pyrethroid. Densities of European red mite, Panonychus ulmi (Koch), were highest in all plots treated with dimethoate and in pyrethroid plots not yet inoculated with New Zealand T. pyri. Densities of apple rust mite, Aculus schlechtendali (Nalepa), and of the stigmaeid predator Zetzellia mali (Ewing) were highest in plots treated with dimethoate and were nearly absent in the IPM plots. Densities of T. pyri were high enough for effective biocontrol in the IPM plots and in the pyrethroid plots 1-2 yr after release of the New Zealand strain, provided pyrethroid was applied just before the resistant strain was released in the orchard. A recurring theme of this study was the generally negative association between densities of phytophagous mites and those of T. pyri, suggesting the ability of this predator to suppress their prey. In contrast, the positive association between phytophagous mites and Z. mali suggests the inability of this predator to regulate their prey at least under the conditions of this study.