Unique Hepatic Cytosolic Arginase Evolved Independently in Ureogenic Freshwater Air-Breathing Teleost,Heteropneustes fossilis

Hepatic cytosolic arginase (ARG I), an enzyme of the urea cycle operating in the liver of ureotelic animals, is reported to be present in an ammoniotelic freshwater air-breathing teleost, Heteropneustes fossilis which has ureogenic potential. Antibodies available against mammalian ARG I showed no cross reactivity with the H. fossilis ARG I. We purified unique ARG I from H. fossilis liver. Purified ARG I is a homotrimer with molecular mass 75 kDa and subunit molecular mass of 24 kDa. The pI value of the enzyme was 8.5. It showed maximum activity at pH 10.5 and 55°C. The Km of purified enzyme for L-arginine was 2.65±0.39 mM. L-ornithine and N^ω-hydroxy-L-arginine showed inhibition of the ARG I activity, with Ki values 0.52±0.02mM and 0.08±0.006mM, respectively. Antibody raised against the purified fish liver ARG I showed exclusive specificity, and has no cross reactivity against fish liver ARG II and mammalian liver ARG I and ARG II. We found another isoform of arginase bound to the outer membrane of the mitochondria which was released by 150–200 mM KCl in the extraction medium. This isoform was immunologically different from the soluble cytosolic and mitochondrial arginase. The results of present study support that hepatic cytosolic arginase evolved in this ureogenic freshwater teleost, H. fossilis. Phylogenetic analysis confirms an independent evolution event that occurred much after the evolution of the cytosolic arginase of ureotelic vertebrates.     

Does fish represent an intermediate stage in the evolution of ureotelic cytosolic arginase I?

Arginase catalyses the last step of the urea cycle. At least two isoenzymes of arginase are known; cytosolic ARG I and mitochondrial ARG II. ARG I is predominantly expressed in liver cytosol, as a part of urea cycle in ureotelic animals. The second isoform ARG II is primarily responsible for non-ureogenic functions, expressed in mitochondria of both hepatic and non-hepatic tissues in most vertebrates. Most micro-organisms and invertebrates are known to have only one type of arginase, whose function is unrelated to ornithine-urea cycle (OUC). However, in ureo-osmotic marine elasmobranchs arginase is localized in liver mitochondria as a part of OUC to synthesize urea for osmoregulation. An evolutionary transition occurred in arginase enzyme in terrestrial ureotelic vertebrates, with the evolution of ARG I from a pre-existing ancestral mitochondrial ARG II. This cytosolic ARG I activity is supposed to have first appeared in lung fishes, but the 40% and 60% distribution of arginase I and II activity in liver and kidney tissue of Heteropneustes fossilis indicates reconsideration of the above fact.     

Alterations in Digestive Enzymes of Three Freshwater Teleostean Fishes by Almix Herbicide: A Comparative Study

Three freshwater teleostean fishes viz., Anabas testudineus (Bloch), Heteropneustes fossilis (Bloch) and Oreochromis niloticus (Linnaeus) were exposed to almix (66.67 mg/l) herbicide for 30 days to investigate the activity of digestive enzymes (amylase, lipase and protease) in stomach, intestine and liver. Amylase activity showed significantly high (p < 0.05) in all the fishes compared to control value and highest activity was observed in liver of A. testudineus (721.99 %) and minimum in intestine of H. fossilis (195.37 %). Lipase activity was also significantly increased (p < 0.05) in all the tissues; but highest in intestine of O. niloticus (235.51 %) and minimum in intestine of A. testudineus (130.51 %). Protease activity also showed similar trends of enhancement; it was maximum in stomach of O. niloticus (362.69 %), whereas in liver of H. fossilis it was rather less (173.72 %). Increased activity of digestive enzymes resulting from tissue damage ultimately affected the fish health due to impairment of digestive physiology confirming the herbicidal contamination on fish species. The sensitivity to the almix herbicide was pronounced in the order of O. niloticus > A. testudineus > H. fossilis.     

Cardiolipin deficiency causes triacylglycerol accumulation in <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

In yeast, the synthesis of cardiolipin (CL) and phosphatidylethanolamine (PE) occurs mainly in mitochondria. CL and PE have overlapping functions, and they are required for mitochondrial function. PE is physiologically linked with triacylglycerol (TAG) metabolism in Saccharomyces cerevisiae, involving an acyl-CoA-independent pathway through the phospholipid:diacylglycerol acyltransferase activity of the Lro1 protein. There is no report on the physiological link between CL and TAG metabolism. Here we report a metabolic link between CL and TAG accumulation in the S. cerevisiae. Our data indicated that CL deficiency causes TAG accumulation, involving an acyl-CoA-dependent pathway through the diacylglycerol acyltransferase activity of the Dga1 protein with no changes in the TAG molecular species. The DGA1 gene deletion from the CL-deficient strains reduced the TAG levels. Data from in vitro and in vivo analyses showed that CL did not affect the enzymatic activity of Dga1. Our data also showed that CL deficiency leads to the up-regulation of acetyl-CoA synthetase genes (ACS1 and ACS2) of the cytosolic pyruvate dehydrogenase bypass pathway. This study establishes a physiological link between CL and TAG metabolism in S. cerevisiae.     

Contribution of arginase to manganese metabolism of <Emphasis Type="Italic">Aspergillus niger</Emphasis>

Aspects of manganese metabolism during normal and acidogenic growth of Aspergillus niger were explored. Arginase from this fungus was a Mn[II]-enzyme. The contribution of the arginase protein towards A. niger manganese metabolism was investigated using arginase knockout (D-42) and arginase over-expressing (ΔXCA-29) strains of A. niger NCIM 565. The Mn[II] contents of various mycelial fractions were found in the order: D-42 strain < parent strain < ΔXCA-29 strain. While the soluble fraction forms 60 % of the total mycelial Mn[II] content, arginase accounted for a significant fraction of this soluble Mn[II] pool. Changes in the arginase levels affected the absolute mycelial Mn[II] content but not its distribution in the various mycelial fractions. The A. niger mycelia harvested from acidogenic growth media contain substantially less Mn[II] as compared to those from normal growth media. Nevertheless, acidogenic mycelia harbor considerable Mn[II] levels and a functional arginase. Altered levels of mycelial arginase protein did not significantly influence citric acid production. The relevance of arginase to cellular Mn[II] pool and homeostasis was evaluated and the results suggest that arginase regulation could occur via manganese availability.     

Recombinant phospholipase A<Subscript>1</Subscript> of the outer membrane of psychrotrophic <Emphasis Type="Italic">Yersinia pseudotuberculosis</Emphasis>: Expression,purification, and characterization

The pldA gene encoding membrane-bound phospholipase A₁ of Yersinia pseudotuberculosis was cloned and expressed in Escherichia coli cells. Recombinant phospholipase A₁ (rPldA) was isolated from inclusion bodies dissolved in 8 M urea by two-stage chromatography (ion-exchange and gel-filtration chromatography) as an inactive monomer. The molecular mass of the rPldA determined by MALDI-TOF MS was 31.7 ± 0.4 kDa. The highly purified rPldA was refolded by 10-fold dilution with buffer containing 10 mM Triton X-100 and subsequent incubation at room temperature for 16 h. The refolded rPldA hydrolyzed 1,2-dioleoyl-sn-glycero-3-phosphatidylcholine in the presence of calcium ions. The enzyme exhibited maximal activity at 37°C and nearly 40% of maximal activity at 15°C. The phospholipase A₁ was active over a wide range of pH from 4 to 11, exhibiting maximal activity at pH 10. Spatial structure models of the monomer and the dimer of Y. pseudotuberculosis phospholipase A₁ were constructed, and functionally important amino acid residues of the enzyme were determined. Structural differences between phospholipases A₁ from Y. pseudotuberculosis and E. coli, which can affect the functional activity of the enzyme, were revealed.     

Procedure for purification of recombinant preMsk1p from <Emphasis Type="Italic">E. coli</Emphasis> determines its properties as a factor of tRNA import into yeast mitochondria

Mitochondrial genomes of many eukaryotic organisms do not code for the full tRNA set necessary for organellar translation. Missing tRNA species are imported from the cytosol. In particular, one out of two cytosolic lysine tRNAs of the yeast Saccharomyces cerevisiae is partially internalized by mitochondria. The key protein factor of this process is the precursor of mitochondrial lysyl-tRNA synthetase, preMsk1p. In this work, we show that recombinant preMsk1p purified from E. coli in native conditions, when used in an in vitro tRNA import system, demonstrates some properties different from those shown by the renatured protein purified from E. coli in the denatured state. We also discuss the possible mechanistic reasons for this phenomenon.     

The obtainment and characteristics of <Emphasis Type="Italic">Kalanchoe pinnata</Emphasis> L. plants expressing the artificial gene of the cecropin P1 antimicrobial peptide

Kalanchoe pinnata L. plants bearing an artificial CP1 gene encoding the cecropin P1 antimicrobial peptide have been obtained. The presence of the CP1 gene in the plant genome has been confirmed by PCR. Cecropin P1 synthesis in transgenic plants has been shown by MALDI mass spectrometry and Western blotting. The obtained plants have been highly resistant to bacterial and fungal phytopathogens, and their extracts have demonstrated antimicrobial activity towards human and animal pathogens. It has been shown that transgenic plants bearing the CP1 gene can be colonized by the beneficial associative microorganisms Methylovorus mays.     

Nuclear mtDNA pseudogenes as a source of new variants of the mtDNA cytochrome <Emphasis Type="Italic">b</Emphasis> haplotypes: A case study of Siberian rubythroat <Emphasis Type="Italic">Luscinia calliope</Emphasis> (Muscicapidae,Aves)

Sequence polymorphism of the mitochondrial DNA cytochrome b gene fragment was analyzed in 21 specimens of subspecies Luscinia calliope calliope (Pallas, 1776) and two specimens of L. c. anadyrensis (Portenko, 1939). On sequence chromatograms, in 19 specimens of L. c. calliope, double peaks of heteroplasmy type in the taxon-specific positions were revealed. Moreover, two clone variants were identified. The first variant was the calliope mitochondrial cyt b gene and the second was the nuclear cyt b pseudogene, similar to the mitochondrial haplotype anadyrensis-camtschatkensis. In L. c. anadyrensis, four clone variants, represented by the mitochondrial calliope and anadyrensis-camtschatkensis cyt b genes and nuclear calliope and sachalinensis cyt b pseudogenes, were identified. Some nuclear cyt b pseudogenes were highly similar (98–99%) to the mitochondrial genes of the subspecies L. c. anadyrensis, L. c. camtschatkensis, and L. c. sachalinensis. In the same time, the majority of nuclear pseudogene sequences were characterized by a high level of polymorphism, caused by nonsynonymous substitutions (up to five substitutions per sequence), the presence of indels in some of the clones, and TAA and TGA stop codons. In our opinion, the mitochondrial haplotypes anadyrensis-camtschatkensis and sachalinensis occurred as a result of intergenomic homologous recombination. This finding provides a new insight into the colonization history of the northeastern part of the range by L. calliope, according to which populating the territory of Chukotka, Kamchatka, and Sakhalin took place at different times and along the independent pathways.     

Study of the Accumulation of Rec A from <Emphasis Type="Italic">Bacillus subtilis</Emphasis> in the Mitochondria of a Recombinant Strain of the Yeast <Emphasis Type="Italic">Yarovia lipolytica</Emphasis>

No eukaryotic species has a system for homologous DNA recombination of the mitochondrial genome. We report on an integrative genetic system based on the pQ-SRUS construct that allows the expression of the RecA recombinase from Bacillus subtilis and its transportation to mitochondria of Yarrowia lipolytica. The targeting of recombinant RecA to mitochondria is provided by leader sequences (5'-UTR and 3'-UTR) derived from the SOD2 gene mRNA, which exhibit affinity to the outer mitochondrial membrane and provides cotranslational import of RecA to the inner space of mitochondria. The accumulation of RecA in mitochondria of the Y. lipolytica recombinant strain bearing the pQ-SRUS construct has been shown by immunoblotting of purified mitochondrial preparations.     

<Emphasis Type="Italic">In vitro</Emphasis> propagation of endangered <Emphasis Type="Italic">Mammillaria</Emphasis> genus (Cactaceae) species and genetic stability assessment using SSR markers

In vitro propagation protocols were established for endangered species of cacti Mammillaria hernandezii, M. dixanthocentron, and M. lanata. In vitro-germinated seedlings were used as the explant source. Three explant types were evaluated as apical, basal, and lateral stem sections. Shoot multiplication was achieved using Murashige and Skoog (MS) medium supplemented with benzyladenine, kinetin, meta-topolin, and thidiazuron in equimolar concentrations (0.0, 0.4, 1.1, 2.2, 4.4, and 8.9 μM). Shoot regeneration was obtained primarily in the lateral stem section explants. In M. hernandezii, an average of 7.4 shoots was regenerated in MS medium with 2.2 μM meta-topolin. M. dixanthocentron and M. lanata averaged 16.7 and 17.9 shoots/explant, respectively, in MS medium supplemented with 1.1 μM meta-topolin. Rooting occurred in MS medium without growth regulators. Three in vitro culture cycles were performed to validate the propagation protocols and to verify genetic stability. Shoots were collected in each cycle and genomic DNA was extracted. Amplified microsatellites were used to compare each genotype with its respective donor plant. Polymorphic information content analysis showed low levels of intra-clonal polymorphisms—M. hernandezii 0.04 and M. dixanthocentron and M. lanata both 0.12. More than 95% of the plants were successfully acclimatized in the greenhouse. After 12 months, plants of M. hernandezii reached the flowering stage; M. dixanthocentron and M. lanata flowered at 24 mo.     

Post-embryonic development of the Malpighian tubules in <Emphasis Type="Italic">Apis mellifera</Emphasis> (Hymenoptera) workers: morphology,remodeling, apoptosis,and cell proliferation

The honeybee Apis mellifera has ecological and economic importance; however, it experiences a population decline, perhaps due to exposure to toxic compounds, which are excreted by Malpighian tubules. During metamorphosis of A. mellifera, the Malpighian tubules degenerate and are formed de novo. The objective of this work was to verify the cellular events of the Malpighian tubule renewal in the metamorphosis, which are the gradual steps of cell remodeling, determining different cell types and their roles in the excretory activity in A. mellifera. Immunofluorescence and ultrastructural analyses showed that the cells of the larval Malpighian tubules degenerate by apoptosis and autophagy, and the new Malpighian tubules are formed by cell proliferation. The ultrastructure of the cells in the Malpighian tubules suggest that cellular remodeling only occurs from dark-brown-eyed pupae, indicating the onset of excretion activity in pupal Malpighian tubules. In adult forager workers, two cell types occur in the Malpighian tubules, one with ultrastructural features (abundance of mitochondria, vacuoles, microvilli, and narrow basal labyrinth) for primary urine production and another cell type with dilated basal labyrinth, long microvilli, and absence of spherocrystals, which suggest a role in primary urine re-absorpotion. This study suggests that during the metamorphosis, Malpighian tubules are non-functional until the light-brown-eyed pupae, indicating that A. mellifera may be more vulnerable to toxic compounds at early pupal stages. In addition, cell ultrastructure suggests that the Malpighian tubules may be functional from dark-brown-eyed pupae and acquire greater complexity in the forager worker bee.     

Isolation and Characterisation of Insulin-Releasing Compounds from <Emphasis Type="Italic">Pseudechis australis</Emphasis> and <Emphasis Type="Italic">Pseudechis butleri</Emphasis> Venom

Crude venom from two elapid snakes Pseudechis australis and Pseudechis butleri was fractionated by gel filtration chromatography and selected fractions screened for in vitro insulin-releasing activity using clonal pancreatic BRIN-BD11 cells. Following acute 20-min incubation at 5.6 mM glucose, 9 fractions exhibited significant (P < 0.001) insulin-releasing activity. Structural characterisation of active fractions was achieved primarily using MALDI–TOF MS and N-terminal Edman degradation sequencing. The partial N-terminal sequences are reported for a total of 7 venom components. Their homology to existing sequences as determined using BLAST searching uncovered the main insulin-releasing families as being phospholipases A₂ and short α-neurotoxins. A number of sequences are reported for the first time from P. butleri venom which is much less studied than the related P. australis.     

Long-term dietary restriction up-regulates activity and expression of renal arginase II in aging mice

Arginase II is a mitochondrial enzyme that catalyses the hydrolysis of L-arginine into urea and ornithine. It is present in other extra-hepatic tissues that lack urea cycle. Therefore, it is plausible that arginase II has a physiological role other than urea cycle which includes polyamine, proline, glutamate synthesis and regulation of nitric oxide production. The high expression of arginase II in kidney, among extrahepatic tissues, might have an important role associated with kidney functions. The present study is aimed to determine the age-associated alteration in the activity and expression of arginase II in the kidney of mice of different ages. The effect of dietary restriction to modulate the age-dependent changes of arginase II was also studied. Results showed that renal arginase II activity declines significantly with the progression of age (p<0.01 and p<0.001 in 6- and 18-month-old mice, respectively as compared to 2-month old mice) and is due to the reduction in its protein as well as the mRNA level (p<0.001 in both 6- and 18-month-old mice as compared to 2-month-old mice). Long-term dietary restriction for three months has significantly up-regulated arginase II activity and expression level in both 2- and 18-month-old mice (p<0.01 and p<0.001, respectively as compared to AL group). These findings clearly indicate that the reducing level of arginase II during aging might have an impact on the declining renal functions. This age-dependent down-regulation of arginase II in the kidney can be attenuated by dietary restriction which may help in the maintenance of such functions.     

Genetic system for maintaining the mitochondrial human genome in yeast <Emphasis Type="Italic">Yarrowia lipolytica</Emphasis>

For the first time, the possibility of maintaining an intact human mitochondrial genome in a heterologous system in the mitochondria of yeast Yarrowia lipolytica is shown. A method for introducing directional changes into the structure of the mitochondrial human genome replicating in Y. lipolytica by an artificially induced ability of yeast mitochondria for homologous recombination is proposed. A method of introducing and using phenotypic selection markers for the presence or absence of defects in genes tRNA-Lys and tRNA-Leu of the mitochondrial genome is developed. The proposed system can be used to correct harmful mutations of the human mitochondrial genome associated with mitochondrial diseases and for preparative amplification of intact mitochondrial DNA with an adjusted sequence in yeast cells. The applicability of the new system for the correction of mutations in the genes of Lys- and Leu-specific tRNAs of the human mitochondrial genome associated with serious and widespread human mitochondrial diseases such as myoclonic epilepsy with lactic acidosis (MELAS) and myoclonic epilepsy with ragged-red fibers (MERRF) is shown.     

Cox2A/Cox2B subunit interaction in <Emphasis Type="Italic">Polytomella</Emphasis> sp. cytochrome <Emphasis Type="Italic">c</Emphasis> oxidase: role of the Cox2B subunit extension

Subunit II of cytochrome c oxidase (Cox2) is usually encoded in the mitochondrial genome, synthesized in the organelle, inserted co-translationally into the inner mitochondrial membrane, and assembled into the respiratory complex. In chlorophycean algae however, the cox2 gene was split into the cox2a and cox2b genes, and in some algal species like Chlamydomonas reinhardtii and Polytomella sp. both fragmented genes migrated to the nucleus. The corresponding Cox2A and Cox2B subunits are imported into mitochondria forming a heterodimeric Cox2 subunit. When comparing the sequences of chlorophycean Cox2A and Cox2B proteins with orthodox Cox2 subunits, a C-terminal extension in Cox2A and an N-terminal extension in Cox2B were identified. It was proposed that these extensions favor the Cox2A/Cox2B interaction. In vitro studies carried out in this work suggest that the removal of the Cox2B extension only partially affects binding of Cox2B to Cox2A. We conclude that this extension is dispensable, but when present it weakly reinforces the Cox2A/Cox2B interaction.     

Rapid MALDI biotyper-based identification and cluster analysis of <Emphasis Type="Italic">Streptococcus iniae</Emphasis>

Streptococcus iniae causes severe mortalities among cultured marine species, especially in the olive flounder (Paralichthys olivaceus), which is economically important in Korea and Japan. Recently, there has been growing concern regarding the emergence of S. iniae as a zoonotic pathogen. Here, 89 S. iniae isolates obtained from diseased olive flounders collected from 2003 to 2008 in Jeju Island, South Korea, were characterized using matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS). The results were aligned both with the available Bruker Daltonics data-base and with a new set of S. iniae data entries developed in our laboratory, and the results were compared. When we used the Bruker Daltonics database, the 89 isolates yielded either “no reliable identification” or were incorrectly identified as Streptococcus pyogenes at the genus level. When we used the new data entries from our laboratory, in contrast, all of the isolates were correctly identified as S. iniae at the genus (100%) and species (96.6%) levels. We performed proteomic analysis, divided the 89 isolates into cluster I (51.7%), cluster II (20.2%), and cluster III (28.1%), and then used the MALDI Biotyper software to identify specific mass peaks that enabled discrimination between clusters and between Streptococcus species. Our results suggest that the use of MALDI TOF MS could outperform the conventional methods, proving easier, faster, cheaper and more efficient in properly identifying S. iniae. This strategy could facilitate the epidemiological and taxonomical study of this important fish pathogen.     

Characterization of chickpea (<Emphasis Type="Italic">Cicer arietinum</Emphasis> L.) lectin for biological activity

Lectins are proteins that are subject of intense investigations. Information on lectin from chickpea (Cicer arietinum L.) with respect to its biological activities are very limited. In this study, we purified lectin from the seeds of chickpea employing DEAE-cellulose and SP-Sephadex ion exchange chromatography and identified its molecular subunit mass as 35 kDa. The free radical scavenging activity of lectin measured by the DPPH assay has IC₅₀ of 0.88 µg/mL. Lectin exerted antifungal activity against Candida krusei, Fusarium oxysporium oxysporium, Saccharomyces cerevisiae and Candida albicans, while antibacterial activity against E. coli, B. subtilis, S. marcescens and P. aeruginosa. The minimum inhibitory concentrations were 200, 240, 160 and 140 µg for C. krusei, F. oxysporium, S. cerevisiae and C. albicans respectively. Lectin was further examined for its antiproliferative potential against cancerous cell line. The cell viability assay indicated a high inhibition activity on Ishikawa, HepG2, MCF-7 and MDA-MB-231 with IC₅₀ value of 46.67, 44.20, 53.58 and 37.46?µg/mL respectively. These results can provide a background for future research into the benefits of chickpea lectin to pharmacological perspective.     

Effect of quinocitrinines from the fungus <Emphasis Type="Italic">Penicillium citrinum</Emphasis> on the respiration of yeasts and bacteria

We studied the effect of quinocitrinines on the respiratory activity of yeasts (Yarrowia lipolytica) and bacteria (Arthrobacter globiformis). Quinocitrinines were shown to activate respiration of native cells in both types of organisms. Studies of yeast mitochondria showed that quinocitrinine exerts an uncoupling effect on oxidative phosphorylation, which activates the respiration, reduces the respiratory control, and decreases the ADP/O ratio. Experiments with intact mitochondria and native cells of Arthrobacter globiformis revealed that quinocitrinine decreases the membrane potential. The uncoupling effect likely constitutes a mechanism of the antibiotic activity of quinocitrinines.