Endonuclease III and endonuclease VIII conditionally targeted into mitochondria enhance mitochondrial DNA repair and cell survival following oxidative stress

Mitochondrial DNA (mtDNA) is exposed to reactive oxygen species (ROS) produced during oxidative phosphorylation. Accumulation of several kinds of oxidative lesions, including oxidized pyrimidines, in mtDNA may lead to structural genomic alterations, mitochondrial dysfunction and associated degenerative diseases. In Escherichia coli, oxidative pyrimidines are repaired by endonuclease III (EndoIII) and endonuclease VIII (EndoVIII). To determine whether the overexpression of two bacterial glycosylase/AP lyases which predominantly remove oxidized pyrimidines from DNA, could improve mtDNA repair and cell survival, we constructed vectors containing sequences for the EndoIII and EndoVIII downstream of the mitochondrial targeting sequence (MTS) from manganese superoxide dismutase (MnSOD) and placed them under the control of the tetracycline (Tet)-response element. Successful integrations of MTS–EndoIII or MTS–EndoVIII into the HeLa Tet-On genome were confirmed by Southern blot. Western blots of mitochondrial extracts from MTS–EndoIII and MTS–EndoVIII clones revealed that the recombinant proteins are targeted into mitochondria and their expressions are doxycycline (Dox) dependent. Enzyme activity assays and mtDNA repair studies showed that the Dox-dependent expressions of MTS–EndoIII and MTS–EndoVIII are functional, and both MTS–EndoIII and MTS–EndoVIII (Dox+) clones were significantly more proficient at repair of oxidative damage in their mtDNA. This enhanced repair led to increased cellular resistance to oxidative stress.     

A voltage-gated K(+) current in renal inner medullary collecting duct cells

We studied the K⁺-selective conductances in primary cultures of rat renal inner medullary collecting duct (IMCD) using perforated-patch and conventional whole cell techniques. Depolarizations above –20 mV induced a time-dependent outward K⁺ current (I_vto) similar to a delayed rectifier. I_vto showed a half-maximal activation around 5.6 mV with a slope factor of 6.8 mV. Its K⁺/Na⁺ selectivity ratio was 11.7. It was inhibited by tetraethylammonium, quinidine, 4-aminopyridine, and Ba²⁺ and was not Ca²⁺ dependent. The delayed rectifying characteristics of I_vto prompted us to screen the expression of Kv1 and Kv3 families by RT-PCR. Analysis of RNA isolated from cell cultures revealed the presence of three Kv -subunits (Kv1.1, Kv1.3, and Kv1.6). Western blot analysis with Kv -subunit antibodies for Kv1.1 and Kv1.3 showed labeling of 70-kDa proteins from inner medulla plasmatic and microsome membranes. Immunocytochemical analysis of cell culture and kidney inner medulla showed that Kv1.3 is colocalized with the Na⁺-K⁺-ATPase at the basolateral membrane, although it is also in the cytoplasm. This is the first evidence of recording, protein expression, and localization of a voltage-gated Kv1 in the kidney IMCD cells. kidney; Kv1.3; potassium channel; potassium transport; whole cell clamp; immunocytochemistry; confocal microscopy     

Ecdysone 20-monooxygenase activity of cytochrome P450 in plants and cell culture of Ajuga reptans L

The concentration of cytochrome P450 and ecdysone 20-monooxygenase activity in plants and callus cell culture of the bugleweed Ajuga reptans L. were determined. The maximal ecdysone 20-monooxygenase activity of cytochrome P450 was found in vegetative rosettes of intact plants. During the stage of flowering, the ecdysone 20-monooxygenase activity of cytochrome P450 in plant leaves was higher than in other organs. It was demonstrated that the content of ecdysteroids in callus cell culture is higher than in the intact plant with concurrent retention of a high ecdysone-20-monooxygenase activity.     

Electron microscopy of basophilic structures of some invertebrate oocytes. I. Periodic lamellane and the nuclear envelope

Highly basophilic plate-shaped regions from oocytes of the surf clam have been examined with the electron microscope. The regions are composed of flat, hollow vesicles perforated by pores arranged, in surface view, in a hexagonal pattern. Cross-sections of this structure show a periodicity consisting of loops (cross-sections of the continuous space within the vesicle) alternating with spaces partly filled with dense material (pores). These structures are shown to resemble closely, the nuclear envelope. Similarities to and differences from basophilic regions of other cells are discussed and it is suggested that the small granules of Palade (38) are represented by granules composing the walls of the annuli of the nuclear envelope and assumed to be present in the annuli of the vesicles. Because of differences in the structure of these regions from basophilic regions of other cells, the name periodic lamellae is suggested since the structures show periodically repeating substructures (annuli) in both cross-sections and surface views.