Peptide conjugates for chromosomal gene targeting by triplex-forming oligonucleotides

Triplex-forming oligonucleotides (TFOs) are DNA-binding molecules, which offer the potential to selectively modulate gene expression. However, the biological activity of TFOs as potential antigene compounds has been limited by cellular uptake. Here, we investigate the effect of cell-penetrating peptides on the biological activity of TFOs as measured in an assay for gene-targeted mutagenesis. Using the transport peptide derived from the third helix of the homeodomain of antennapedia (Antp), we tested TFO–peptide conjugates compared with unmodified TFOs. TFOs covalently linked to Antp resulted in a 20-fold increase in mutation frequency when compared with ‘naked’ oligonucleotides. There was no increase above background in mutation frequency when Antp by itself was added to the cells or when Antp was linked to mixed or scrambled sequence control oligonucleotides. In addition, the TFO–peptide conjugates increased the mutation frequency of the target gene, and not the control gene, in a dose-responsive manner. Confocal microscopy using labeled oligonucleotides indicated increased cellular uptake of TFOs when linked to Antp, consistent with the gene-targeting data. These results suggest that peptide conjugation may enhance intranuclear delivery of reagents designed to bind to chromosomal DNA.     

Phycobiliprotein methylation. Effect of the gamma-N-methylasparagine residue on energy transfer in phycocyanin and the phycobilisome

The phycobiliproteins contain a conserved unique modified residue, gamma-N-methylasparagine at beta-72. This study examines the consequences of this methylation for the structure and function of phycocyanin and of phycobilisomes. An assay for the protein asparagine methylase activity was developed using [methyl-3H]S-adenosylmethionine and apophycocyanin purified from Escherichia coli containing the genes for the alpha and beta subunits of phycocyanin from Synechococcus sp. PCC 7002 as substrates. This assay permitted the partial purification, from Synechococcus sp. PCC 6301, of the activity that methylates phycocyanin and allophycocyanin completely at residue beta-72. Using the methylase assay, two independent nitrosoguanidine-induced mutants of Synechococcus sp. PCC 7942 were isolated that do not exhibit detectable phycobiliprotein methylase activity. These mutants, designated pcm 1 and pcm 2, produce phycocyanin and allophycocyanin unmethylated at beta-72. The phycobiliproteins in these mutants are assembled into phycobilisomes and can be methylated in vitro by the partially purified methylase from Synechococcus sp. PCC 6301. The mutants produce phycobiliproteins in amounts comparable to those of wild-type and the mutant and wild-type phycocyanins are equivalent with respect to thermal stability profiles. Monomeric phycocyanins purified from these strains show small spectral shifts that correlate with the level of methylation. Phycobilisomes from the mutant strains exhibit defects in energy transfer, both in vivo and in vitro, that are also correlated with deficiencies in methylation. Unmethylated or undermethylated phycobilisomes show greater emission from phycocyanin and allophycocyanin and lower fluorescence emission quantum yields than do fully methylated particles. The results support the conclusion that the site-specific methylation of phycobiliproteins contributes significantly to the efficiency of directional energy transfer in the phycobilisome.     

Fluorescence-based assay for reactive oxygen species: a protective role for creatinine

Attack by reactive oxygen species leads to a decay in phycoerythrin fluorescence emission. This phenomenon provides a versatile new assay for small molecules and macromolecules that can function as protective compounds. With 1-2 x 10(-8) M phycoerythrin, under conditions where peroxyl radical generation is rate-limiting, the fluorescence decay follows apparent zero-order kinetics. On reaction with HO., generated with the ascorbate-Cu2+ system, the fluorescence decays with apparent first-order kinetics. Examination of the major components of human urine in this assay confirms that at physiological concentrations, urate protects against both types of oxygen radicals. A novel finding is that creatinine protects efficiently by a chelation mechanism against radical damage in the ascorbate-Cu2+ system at creatinine, ascorbate, and Cu2+ concentrations comparable to those in normal urine. Urate and creatinine provide complementary modes of protection against reactive oxygen species in the urinary tract.     

Migratory behaviour and desiccation tolerance of protostrongylid nematode first-stage larvae

Migration of first-stage larvae (L1) from faeces to soil is a crucial stage in the life-history of protostrongylids transmitted via land snails. Migration of Muellerius cf. capillaris and a Cystocaulus sp. L1 from fresh Nubian ibex (Capra ibex nubiana) faeces (48–50% water content, W.C.) to substrate soils (at 100% r.h., 26°C) was measured experimentally using dry (3 ± 1% W.C.), wet (31 ± 0.43% W.C.) and flooded (48.4 ± 2.45% W.C.) soils. The highest migration rates (90.4 ± 1.6% migration) in both species occurred on flooded soils when the faecal pellet W.C. reached 90%. The next highest migration rates (43.2 ± 3.6% migration, at 60% faecal W.C.) were on the wet soils and no migration occurred on dry soil or dry-substrate papers. Migration rates did not differ significantly (P > 0.05) between species. Active Theba pisana were not infected by M. cf. capillaris L1 on dry infested soils, but were infected following rehydration of the same soils. By day 10, L1 of M. cf. capillaris demonstrated lower survival rates in water and in 97% and 76% r.h. (74.5%, 15.2% and 1.9%, respectively) than the Cystocaulus sp. (97.5%, 43.8%, 43.3%) and Protostrongylus sp. (97.9%, 43.2%, 23.8%, P < 0.05). All three nematodes had a remarkably high survival rate (> 99% overall survival, by day 10) when exposed directly to 0% r.h. at 23°C, Results demonstrate the ability of L1 to survive extreme desiccation through anhydrobiosis. Migration of L1 from facces to soil can take place only during rains which coincide, with peak activity of land snails in desert habitat.     

Implications phylogénétiques de I'ultrastructure de la spermatogenèse, du spermatozoïde et de I'ovogenèse du TurbellariéUrastoma cyprinae ("Prolecithophora", Urastomidae)

Spermiogenesis in Urastoma cyprinae (Graff. 1882) involvcs a progressive lengthening of the spermatid. Free flagella are only transitory. The mature spermatozoon is fusibrm. 45μm in length and 2.5 μm in width. It contains two incorporated axonermes of the flatworm 9+"1" pattern, two elongated mitochondria. an elongated nucleus and a row or cortical longitudinal microtubules. Observations on oogenesis concerncd only the immuturc ovary. lmmature oocytes contain few dense granules and accssory cells were not ohserved. Phylogenetic implications of a biflagellate spermatozoon in a Prolecithophora are important. The presence of two 9 +"1" axonemes confirms that Urastoma (and the Prolecithophora) belongs to the taxon Trepaxonemata Ehlers. Previous electron microscope studies on spermitozoa of Prolecithophora (four genera) only dealt with aflagellate spermatozoa. On this basis, Ehlers (1985) proposed two autapomorphics for the taxon Prolecithophora: aflagellate spermatozoon and spermatozoal mitochondrial derivatives with abundant membranes. The present observations on Urastoma contradict these two autapomorphics. The taxon Prolecithophora cannot be defined by autapomorphies of the spermatozoon.     

The effect of temperature and relative humidity on the formation of Metarhizium anisopliae chlamydospores in tick eggs

The influence of ambient conditions on the development of Metarhizium anisopliae chlamydospores in tick eggs is reported for the first time. The infection of tick eggs by M. anisopliae involves common events, such as adhesion, conidial germination, appressoria formation, invasion, and development within the eggs. However, the final stage of fungal development differs according to the environmental conditions. At high humidity (close to 100%) and moderate temperature (25°C) the fungus emerged from the eggs and formed conidiophores and conidia externally on the dead eggs. Elevating the temperature to 30°C or reducing humidity to 55-75% induced the production of chlamydospores inside the eggs, without conidiogenesis. When eggs with mature chlamydospores were returned to the appropriate conditions (25°C and 100% RH), conidiogenesis was recovered. Formation of chlamydospores, observed by means of histology and TEM, began with the thickening and septation of hyphae. As the chlamydospore wall thickened a new external undulated wall layer appeared. The mature chlamydospore in eggs has an oval shape (5.3 ± 0.9 microm long, 2.5 ± 0.2 microm wide); its wall comprises three distinct layers. The ability of M. anisopliae to produce chlamydospores under harsh conditions is advantageous and should be considered in application.     

Metarhizium anisopliae conidial responses to lipids from tick cuticle and tick mammalian host surface

Conidial germination and the formation of appressoria are important events in the interactions between entomopathogenic fungi and their arthropod hosts. In this study, we demonstrate the effects of lipids extracted from tick epicuticle and the surface of a mammalian host (calf) on conidial germination and the development of appressoria in two subspecies of Metarhizium anisopliae, M. anisopliae var. anisopliae (M.an.an.-7) and M. anisopliae var. acridum (M.an.ac.-5), which have different levels of virulence toward ticks. Pentane extracts of epicuticles of ticks susceptible and resistant to fungal infection always stimulated the germination of M.an.an.-7 conidia and the development of their appressoria; whereas the effects of dichloromethane (DCM) extracts of tick epicuticle varied depending on the tick. The DCM extracts from most of the tick species and developmental stages stimulated conidial germination and/or the formation of appressoria in M.an.an.-7. However, a DCM extract of lipids from the most resistant tick, engorged Hyalomma excavatum female, inhibited the germination of M.an.an.-7 conidia. Conidia of the non-virulent M.an.ac.-5 did not germinate on agarose amended with any of the examined tick extracts. However, when the tick extracts were placed on bactoagar, conidial germination increased 7- to 8-fold. Extracts from the skin, hair and ear secretions of a calf stimulated conidial germination and the formation of appressoria in M.an.an.-7, but not M.an.ac.-5. This study demonstrates that lipids from tick epicuticles and mammalian skin selectively affect the germination of conidia of entomopathogenic fungi. The effects of these lipids may explain the variability in tick control these fungi provide for different hosts.