Generative and somatic production of female sturgeon at a research farm in Primorsky Krai as a basis for manufacturing gastronomic caviar

The generative and somatic characteristics of female sturgeon of five species and three hybrid forms, which were reared at the TINRO Center’s warm-water cage farm at the north of Primorsky Krai and used in caviar production, are considered. Data on the somatic growth, roe output, functional fecundity, mean egg weight, and gonadosomatic index of the female Amur Sturgeon, Kaluga, Siberian Sturgeon of the Lena and the Baikal populations, Sterlet of the Volga population, hybrids of Russian Sturgeon and Siberian Sturgeon, hybrids of Siberian Sturgeon and Amur Sturgeon, and hybrids of Kaluga and Amur Sturgeon are analyzed. The sexual maturity age and interspawning intervals are defined in the studied females. Domesticated females of all the species are shown to mature a few years earlier than those in natural conditions and to have a greater body weight. The interspawning intervals shorten by a few years in domesticated females. Most female Sterlet and some females from both populations of Siberian Sturgeon spawn annually, while the remainder spawn once in 2 years. The interspawning intervals in two species of sturgeon that inhabit the Amur River and in hybrid forms usually last for 2 years. With age, values of characteristics such as roe amount, fecundity, mean egg weight, and roe output relative to body weight grow in females spawning for the second time in the warm-water farm. The maximum roe output is found in female Kalugas at the second spawning. Then the species follow in the order of decreasing of roe output: Amur Sturgeon, hybrids of Amur Sturgeon with Kaluga, hybrids of Siberian Sturgeon with Amur Sturgeon, hybrids of Russian Sturgeon with Siberian Sturgeon, Siberian Sturgeon of the Baikal population, Siberian Sturgeon of the Lena population, and Sterlet. The hybrid between the Russian Sturgeon and Siberian Sturgeon shows the best processability and survivability characteristics; the Amur Sturgeon and Sterlet follow.     

Transformation of Rickettsia prowazekii to Rifampin Resistance

Rickettsia prowazekii, the causative agent of epidemic typhus, is an obligate intracellular parasitic bacterium that grows directly within the cytoplasm of the eucaryotic host cell. The absence of techniques for genetic manipulation hampers the study of this organism’s unique biology and pathogenic mechanisms. To establish the feasibility of genetic manipulation in this organism, we identified a specific mutation in the rickettsial rpoB gene that confers resistance to rifampin and used it to demonstrate allelic exchange in R. prowazekii. Comparison of the rpoB sequences from the rifampin-sensitive (Rif^s) Madrid E strain and a rifampin-resistant (Rif^r) mutant identified a single point mutation that results in an arginine-to-lysine change at position 546 of the R. prowazekii RNA polymerase β subunit. A plasmid containing this mutation and two additional silent mutations created in codons flanking the Lys-546 codon was introduced into the Rif^s Madrid E strain of R. prowazekii by electroporation, and in the presence of rifampin, resistant rickettsiae were selected. Transformation, via homologous recombination, was demonstrated by DNA sequencing of PCR products containing the three mutations in the Rif^r region of rickettsial rpoB. This is the first successful demonstration of genetic transformation of Rickettsia prowazekii and represents the initial step in the establishment of a genetic system in this obligate intracellular pathogen.     

Involvement of mtDNA damage in free fatty acid-induced apoptosis

A growing body of evidence indicates that free fatty acids (FFA) can have deleterious effects on beta-cells. It has been suggested that the beta-cell dysfunction and death observed in diabetes may involve exaggerated activation of the inducible form of nitric oxide synthase (iNOS) by FFA, with the resultant generation of excess nitric oxide (NO). However, the cellular targets with which NO interact have not been fully identified. We hypothesized that one of these targets might be mitochondrial DNA (mtDNA). Therefore, experiments were initiated to evaluate damage to mtDNA caused by exposure of INS-1 cells to FFA (2/1 oleate/palmetate). The results showed that FFA caused a dose-dependent increase in mtDNA damage. Additionally, using ligation-mediated PCR, we were able to show that the DNA damage pattern at the nucleotide level was identical to the one induced by pure NO and different from damage caused by peroxynitrite or superoxide. Following exposure to FFA, apoptosis was detected by DAPI staining and cytochrome c release. Treatment of INS-1 cells with the iNOS inhibitor aminoguanidine protected these cells from mtDNA damage and diminished the appearance of apoptosis. These studies suggest that mtDNA may be a sensitive target for NO-induced toxicity which may provoke apoptosis in beta-cells following exposure to FFA.     

Contribution of mitochondrial DNA repair to cell resistance from oxidative stress

Numerous studies have revealed that a part of the cellular response to chronic oxidative stress involves increased antioxidant capacity. However, another defense mechanism that has received less attention is DNA repair. Because of the important homeostatic role of mitochondria and the exquisite sensitivity of mitochondrial DNA (mtDNA) to oxidative damage, we hypothesized that mtDNA repair plays an important role in the protection against oxidative stress. To test this hypothesis mtDNA damage and repair was evaluated in normal HA1 Chinese hamster fibroblasts and oxidative stress-resistant variants isolated following chronic exposure to H2O2 or 95% O2. Reactive oxygen species were generated enzymatically using xanthine oxidase and hypoxanthine. When treated with xanthine oxidase reduced levels of initial mtDNA damage and enhanced mtDNA repair were observed in the cells from the oxidative stress-resistant variants, relative to the parental cell line. This enhanced mtDNA repair correlated with an increase in mitochondrial apurinic/apyrimidinic endonuclease activity in both H2O2- and O2-resistant HA1 variants. This is the first report showing enhanced mtDNA repair in the cellular response to chronic oxidative stress. These results provide further evidence for the crucial role that mtDNA repair pathways play in protecting cells against the deleterious effects of reactive oxygen species.     

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.     

Cultivation of Triple Hybrids of Russian,Siberian, and Amur Sturgeons at a Warm-Water Fish Farm

Russian Journal of Marine Biology - This study provides biological, fishery, and production characteristic of spawning females of a triple hybrid between the Russian sturgeon, Siberian sturgeon,...     

Mitochondrial DNA Damage via Augmented Oxidative Stress Regulates Endoplasmic Reticulum Stress and Autophagy: Crosstalk,Links and Signaling

Saturated free fatty acids (FFAs) have been implicated in the increase of oxidative stress, mitochondrial dysfunction, endoplasmic reticulum (ER) stress, autophagy, and insulin resistance (IR) observed in skeletal muscle. Previously, we have shown that palmitate-induced mitochondrial DNA (mtDNA) damage triggers mitochondrial dysfunction, mitochondrial reactive oxygen species (mtROS) production, apoptosis and IR in L6 myotubes. The present study showed that mitochondrial overexpression of human 8-oxoguanine DNA glycosylase/AP lyase (hOGG1) decreased palmitate-induced carbonylation of proteins in mitochondria. Additionally, we found that protection of mtDNA from palmitate-induced damage significantly diminished markers of both ER stress and autophagy in L6 myotubes. Moreover, we observed that the addition of ROS scavenger, N-acetylcystein (NAC), to palmitate diminished both ER stress and autophagy markers mimicking the effect of mitochondrial overexpression of hOGG1. This is the first study to show that mtDNA damage is upstream of palmitate-induced ER stress and autophagy in skeletal muscle cells.     

Conditional targeting of the DNA repair enzyme hOGG1 into mitochondria

Oxidative damage to mitochondrial DNA (mtDNA) has been suggested to be a key factor in the etiologies of many diseases and in the normal process of aging. Although the presence of a repair system to remove this damage has been demonstrated, the mechanisms involved in this repair have not been well defined. In an effort to better understand the physiological role of recombinant 8-oxoguanine DNA glycosylase/apurinic lyase (OGG1) in mtDNA repair, we constructed an expression vector containing the gene for OGG1 downstream of the mitochondrial localization sequence from manganese-superoxide dismutase. This gene construct was placed under the control of a tetracycline-regulated promoter. Transfected cells that conditionally expressed OGG1 in the absence of the tetracycline analogue doxycycline and targeted this recombinant protein to mitochondria were generated. Western blots of mitochondrial extracts from vector- and OGG1-transfected clones with and without doxycycline revealed that removal of doxycycline for 4 days caused an approximate 8-fold increase in the amount of OGG1 protein in mitochondria. Enzyme activity assays and DNA repair studies showed that the doxycycline-dependent recombinant OGG1 is functional. Functional studies revealed that cells containing recombinant OGG1 were more proficient at repairing oxidative damage in their mtDNA, and this increased repair led to increased cellular survival following oxidative stress.