Significant impact of divalent metal ions on the fidelity,sugar selectivity,and drug incorporation efficiency of human PrimPol

Human PrimPol is a recently discovered bifunctional enzyme that displays DNA template-directed primase and polymerase activities. PrimPol has been implicated in nuclear and mitochondrial DNA replication fork progression and restart as well as DNA lesion bypass. Published evidence suggests that PrimPol is a Mn²⁺-dependent enzyme as it shows significantly improved primase and polymerase activities when binding Mn²⁺, rather than Mg²⁺, as a divalent metal ion cofactor. Consistently, our fluorescence anisotropy assays determined that PrimPol binds to a primer/template DNA substrate with affinities of 29 and 979 nM in the presence of Mn²⁺ and Mg²⁺, respectively. Our pre-steady-state kinetic analysis revealed that PrimPol incorporates correct dNTPs with 100-fold higher efficiency with Mn²⁺ than with Mg²⁺. Notably, the substitution fidelity of PrimPol in the presence of Mn²⁺ was determined to be in the range of 3.4 × 10⁻² to 3.8 × 10⁻¹, indicating that PrimPol is an error-prone polymerase. Furthermore, we kinetically determined the sugar selectivity of PrimPol to be 57–1800 with Mn²⁺ and 150–4500 with Mg²⁺, and found that PrimPol was able to incorporate the triphosphates of two anticancer drugs (cytarabine and gemcitabine), but not two antiviral drugs (emtricitabine and lamivudine).     

Synthesis of alkali metal hexahydroaluminate complexes using dimethyl ether as a reaction medium

A novel method of preparation of hexahydroaluminate complexes M₃AlH₆ (M = Li, Na or K) from the corresponding alkali metal hydride and tetrahydroaluminate has been explored, using dimethyl ether (Me₂O) as a solvent at near-ambient temperatures. The results are compared with those obtained using a recently established mechanochemical approach. Characterization of the products by powder X-ray diffraction revealed M₃AlH₆ to be formed in high yield for M = Li and Na, but not for M = K. The attempted preparation of Li₂NaAlH₆ and Li₂KAlH₆ was unsuccessful.     

Anti-fibrillogenic and fibril destabilizing effects of metal ions on cystatin fibrils

Metals such as Cu²⁺, Fe³⁺, and Zn²⁺ are major contributors to the biology of a brain in stages of health, aging, and disease because of their unique effects on both protein structures (misfolding) and oxidative stress. The relationship between metal ions and neurodegenerative diseases is very complicated. Our study highlights how metal ions influence amyloid formation at low pH and on preformed amyloid fibrils. By using thioflavin T assay, ANS fluorescence, Congo red assay, circular dichroism, and microscopy to elucidate the effects of Cu²⁺, Fe³⁺, and Zn²⁺ on goat brain cystatin (GBC) aggregation at low pH. Results showed that Cu²⁺ and Fe³⁺ inhibit fibril formation of GBC by promoting amorphous aggregates. However, Zn²⁺ exclusively promotes fibril formation at low pH, leading to the formation of more ordered aggregates. Furthermore, the combined results of these complementary methods also suggested that Cu²⁺ and Fe³⁺ destabilize the β-sheet secondary structure of preformed amyloid fibrils of GBC.     

Neural inhibition sharpens auditory spatial selectivity of bat inferior collicular neurons

This study examines the role of neural inhibition in auditory spatial selectivity of inferior collicular neurons of the big brown bat, Eptesicus fuscus, using a two-tone inhibition paradigm. Two-tone inhibition decreases auditory spatial response areas but increases the slopes of directional sensitivity curves of inferior collicular neurons. Inferior collicular neurons have either directionally-selective or hemifield directional sensitivity curves. A directionally-selective curve always has a peak which is at least 50% larger than the minimum. A hemifield directional sensitivity curve rises from an ipsilateral angle by more than 50% and either reaches a plateau or declines by less than 50% over a range of contralateral angles. Two-tone inhibition does not change directionally-selective curves but changes most hemifield directional sensitivity curves into directionally-selective curves. Auditory spatial selectivity determined both with and without two-tone inhibition increases with increasing best-excitatory frequency. Sharpening of auditory spatial selectivity by two-tone inhibition is larger for neurons with smaller differences between excitatory and inhibitory best frequencies. The effect of two-tone inhibition on auditory spatial selectivity increases with increasing inhibitory tone intensity but decreases with increasing intertone interval. The implications of these findings in bat echolocation are discussed. Accepted: 18 January 2000     

Alga consumption of four dominant planktonic crustaceans in Lake Balaton (Hungary)

Between 1981–83 the gut contents ofDaphnia galeata, D. cucullata, Eudiaptomus gracilis, andCyclops vicinus were examined with light and scanning electron microscope to obtain information on the feeding of these species in Lake Balaton. The twoDaphnia species feed mainly on abioseston, and it is assumed that their primary nutrient source was organic matter adsorbed onto the surfaces of the abioseston granules plus bacteria and detritus.E. gracilis feeds on algae, showing a preference for green algae and diatoms.C. vicinus is also a prodigious consumer of algae in Lake Balaton, utilizing the whole size spectrum of phytoplankton. Concerning the trophic relationships between phytoplankton and zooplankton in Lake Balaton, that between diatoms and bothE. gracilis andC. vicinus is the most conspicouos. Convincing evidence for an extensive utilization of blue-green algae was not found. Though there is no firm evidence yet, it is likely that theDaphnia are dependent on organic matter adsorbed on the abioseston.     

The host range and selectivity of a parasitic plant: Rhinanthus minor L.

Summary Rhinanthus minor (Yellow-rattle) is a widespread hemiparasitic plant of grassland habitats throughout Britain. Association analysis of the dune vegetation at Holme-next-the-Sea in eastern England revealed only two potential host plants through positive association. In contrast direct examination of the root systems revealed haustorial connections with 20 host species. The number of species parasitized by one plant ranged from one to seven. Data from another four sites in Britain and one in central Europe indicate that the natural host range of R. minor encompasses at least 50 species from 18 families with 22% in the Leguminosae and 30% in the Gramineae. Comparison of the number of haustorial connections made to each species with the abundance of roots in the soil shows that R. minor is a highly selective parasite, but that the selectivity is not consistent between populations or between plants from different parts of the same population. The reasons for host selectivity are discussed.     

Bacterial interactions with silver

Summary This review examines interactions between bacteria and the biologically non-essential metal, silver. Aspects of silver toxicity, tolerance and accumulation (possible binding and uptake as opposed to energy-dependent transport) in bacteria are discussed. In addition, plasmid biology is examined briefly since little information is available on the exact mechanism(s) of plasmid-endoced silver resistance in bacteria.     

Prey selectivity inEuseius tularensis [Acari: Phytoseiidae]

Differences in responsiveness to potential prey and pollen were tested in the facultative predator,Euseius tularensis Congdon. Following isolation without food for 16 h, adult female mites were observed with one of the following potential foods: pollen ofMalephora crocea (Jacquin) Schwantes,Scirtothrips citri (Moulton) 2nd instar larvae,Tetranychus pacificus McGregor nymphs and ♂,Panonychus citri (McGregor) nymphs and ♂,E. hibisci (Chant) larvae, andE. tularensis larvae. Responses were compared using the proportion of encounters that resulted in consumption. This proportion was highest when pollen was encountered, followed byS. citri andT. pacificus. Consumption was much lower in response toP. citri and congeneric larvae. WhenS. citri larvae were encountered a 2nd time, 1 to 2 h after the initial capture and consumption, responsiveness increased to a level equal to the response observed with pollen. These results suggest thatE. tularensis is a more effective biological control agent of citrus thrips (S. citri) than of citrus red mite (P. citri).     

1H NMR investigation of reduced copper-cobalt superoxide dismutase

Human copper-cobalt superoxide dismutase in the reduced form has been investigated through ¹H NMR techniques. The aim is to monitor the structural properties of this derivative and to compare them with those of reduced and oxidized native superoxide dismutases. The observed signals of the cobalt ligands have been assigned as well as the signals of the histidines bound to copper(I). The latter signals experience little pseudocontact shifts which allow a rough orientation of the magnetic susceptibility tensor in the molecular frame. The connectivities indicate that, although the histidine bridge is broken in the reduced form, the interproton distances between ligands of both ions are essentially the same.Abbreviations WEFT water eliminated Fourier transform - NOE nuclear Overhauser effect - NOESY NOE spectroscopy - COSY correlation spectroscopy - TOCSY total correlation spectroscopy - SOD superoxide dismutase - E₂Co(II)SOD SOD with empty copper site (E=empty) and with cobalt(II) in the Zinc(II) site Offprint requests to: I. Bertini