Evolution and Regulation of the Downstream Gene of Hypoxia-Inducible Factor-1α in Naked Carp (Gymnocypris przewalskii) from Lake Qinghai, China

The naked carp (Gymnocypris przewalskii) is a native teleost of Lake Qinghai (altitude, 3.2 km) on the Qinghai-Tibet Plateau in China. Hypoxia-inducible factor (HIF)-1α of Gymnocypris przewalskii was cloned and a phylogenetic tree for vertebrate HIF-1α was constructed. By analysis of maximum likelihood models of codon substitutions for HIF-1α, three positive sites in the branch lineages of crucian carp, eelpout, and flounder, and a higher proportion of neutral sites in naked carp, antarctic eelpout, rainbow trout, and grayling, were detected among all teleosts. It seems that low habitat temperatures relax the purifying selection of HIF-1α in these stenothermal coldwater fish, and both cold and hypoxic lake water contributed to the evolution of the HIF-1α gene in the naked carp. Furthermore, Glut 1 mRNA, a gene downstream from HIF-1α, has a time-course- and tissue-specific dependent response to hypoxic challenge.     

Cloning of Black Carp β-actin Gene and Primarily Detecting the Function of Its Promoter Region

A 3 338 bp DNA fragment including the open reading frame and 5′-flanking region of β-actin gene for black carp genome was obtained through PCR amplification. Analysis of the sequencing results indicated the ORF of black carp β-actin gene encoding a 375 amino acid protein that shares a high degree of conservation to other known actins. The black carp β-actin sequence showed 100% identity to common carp, grass carp, and zebrafish, 99.2% identity to human and Norway rat β-actin gene, 98.9% and 98.1% identity to chicken and Kenyan clawed frog β-actin gene, respectively. The promoter region of black carp β-actin gene was inserted into the promoterless pEGFP₁ vector. The recombinant plasmid was microinjected into the fertilized eggs of mud loach before two-cell stage as well as transfected into HeLa cell line. GFP expression was found in 50% of mud loach embryos and 2/3 HeLa cells. The GFP expression could be observed in every part of the mud loach embryos, and in some embryos, the GFP was expressed in the whole body. Thus, the usefulness of black carp β-actin promoter as a ubiquitous expression promoter was confirmed using the EGFP as a reporter gene.     

Proximate Composition of Freshwater Fish — Grass Carp,Puntius gonionotus and Tilapia

Summary The proximate analyses of freshwater fish — Grass carp (Ctenopharyngodon idellus), Puntius gonionotus and Tilapia (male hybrid) are compared. Dried meals from eviscerated freshwater fish contained 57.36–75.92% protein; 4.48–31.84% fat and 10.68–22.88% ash. The results indicate that Tilapia had a higher fat content than gonionotus and grass carp, the latter having the lowest fat content. The reverse is true for protein, ash and minerals like sodium, phosphorus and potassium. The composition of grass carp feeding on different diets, such as Hydrilla verticillata, Napier grass (Pennisetum purpureum) and tapioca leaves (Manihot utilissins) indicated only a slight difference in protein content but a significant difference in fat and ash. The tabulated results show that grass carp and goniotus were of higher nutritive value when compared to the Tilapia.     

A Tale of Four “Carp”: Invasion Potential and Ecological Niche Modeling

Background

Invasive species are a serious problem in ecosystems, but are difficult to eradicate once established. Predictive methods can be key in determining which areas are of concern regarding invasion by such species to prevent establishment [1]. We assessed the geographic potential of four Eurasian cyprinid fishes (common carp, tench, grass carp, black carp) as invaders in North America via ecological niche modeling (ENM). These “carp” represent four stages of invasion of the continent (a long-established invader with a wide distribution, a long-established invader with a limited distribution, a spreading invader whose distribution is expanding, and a newly introduced potential invader that is not yet established), and as such illustrate the progressive reduction of distributional disequilibrium over the history of species'' invasions.

Methodology/Principal Findings

We used ENM to estimate the potential distributional area for each species in North America using models based on native range distribution data. Environmental data layers for native and introduced ranges were imported from state, national, and international climate and environmental databases. Models were evaluated using independent validation data on native and invaded areas. We calculated omission error for the independent validation data for each species: all native range tests were highly successful (all omission values <7%); invaded-range predictions were predictive for common and grass carp (omission values 8.8 and 19.8%, respectively). Model omission was high for introduced tench populations (54.7%), but the model correctly identified some areas where the species has been successful; distributional predictions for black carp show that large portions of eastern North America are at risk.

Conclusions/Significance

ENMs predicted potential ranges of carp species accurately even in regions where the species have not been present until recently. ENM can forecast species'' potential geographic ranges with reasonable precision and within the short screening time required by proposed U.S. invasive species legislation.     

Purification and Characterization of α1-Proteinase Inhibitor from Carp (Cyprinus carpio) Serum

α₁-Proteinase inhibitor was purified to homogeneity from carp serum with an increase in specific inhibitory activity of 17-fold and a 3% recovery rate. The inhibitor was estimated to have molecular weight of 55,000 under reducing and nonreducing conditions, indicating its composition of a single polypeptide. The inhibitor immunologically crossreacted faintly with carp muscular serine proteinase inhibitor but had no crossreactivity with serine proteinase inhibitors from other species. Carp serum inhibitor exhibited marked stability over broad pH ranges of 4.0 to 10.0 and temperatures below 55°C. The inhibitor potently inhibited the activities of carp intestinal and fish myofibril-binding proteinases, and its respective inhibitions of trypsin-type and carp muscular proteinases were more severe than those of chymotrypsin-type and white croaker muscular proteinases. Its inhibitions were similar to those of bovine pancreatic trypsin and α-chymotrypsin, and the amount required to completely inactivate 0.2 μg of each of these two proteinases was evaluated as 0.43 to 0.45 μg. This indicates a molar ratio close to 1:1 during combination of the inhibitor with each proteinase. In addition, its ability to form irreversible complexes with the proteinases was observed electrophoretically and immunologically under denaturing and reducing conditions. Received April 3, 1998; accepted June 30, 1998.     

Competition among river planktivores: are native planktivores still fewer and skinnier in response to the Silver Carp invasion?

Planktivorous Silver Carp Hypophthalmichthys molitrix and Bighead Carp H. nobilis have successfully invaded much of the Upper Mississippi River System and its tributaries during the last 30 years. During the initial years of the invasion, concurrent declines in the body condition and the catch per unit effort (CPUE) of planktivorous Gizzard Shad Dorosoma cepedianum and Bigmouth Buffalo Ictiobus cyprinellus were attributed to competition with Asian carp. Using an additional seven years of data (2007–2013), we assessed whether Silver Carp have continued to exert adverse pressure on the condition, CPUE, and biomass of native planktivores or whether there is evidence of a potential rebound in the populations of native planktivores. The extended data set reaffirms the body condition and the CPUE of Bigmouth Buffalo remain significantly reduced. However, unlike previous analyses, we also observed significant reductions in the CPUE of Gizzard Shad. Additionally, new results show that the CPUE and biomass of Bigmouth Buffalo were more inversely related to Silver Carp CPUE and biomass relative to the CPUE and biomass of Gizzard Shad. These results reinforce that Silver Carp likely suppress native planktivores and also suggest that diet and other life-history traits may explain some of the subtle differences in species-specific responses. Our results also emphasize that long-term data can be critical to understanding how non-native species can influence native fish population dynamics and how this influence may change over time.     

Biotransformation of Digitoxin in Cell Cultures of Capsicum frutescens in the Presence of β-cyclodextrin

Cell suspension cultures of Capsicum frutescens accumulated digoxin, purpureaglycoside A and other unknown derivatives when digitoxin, a cardiac glycoside, was used as a precursor. The feeding of digitoxin complexed with &#103 -cyclodextrin increased the accumulation of digoxin, purpureaglycoside A and other unknown derivatives. Control cultures (without digitoxin) did not produce any of these metabolites. The growth of cells was affected by both digitoxin as well as digitoxin- &#103 -cyclodextrin. The accumulation of purpureaglycoside A and digoxin reached a maximum of 1241 and 374 &#119 g 100 ml &#109 1 culture on the 6th and 2nd day, respectively, which was 3.9 and 4.5 fold higher than cultures treated with digitoxin alone (sampled on the 13th day). The other unknown derivatives formed in digitoxin- &#103 -cyclodextrin fed cultures were 15 times higher than digitoxin alone fed C. frutescens cultures. The addition of glucose to digitoxin- &#103 -cyclodextrin treated cultures increased the accumulation of purpureaglycoside A which reached a maximum of 3589 &#119 g 100 ml &#109 1 culture after 12 h incubation, which was a 2.9 fold increase over cultures treated with digitoxin- &#103 -cyclodextrin alone.     

Evolutionary Advantages of Participation of Vasopressin instead of Vasotocin in Regulation of Water–Salt Balance in Mammals

In experiments on non-anesthetized Wistar white rats there was studied reaction of kidney to an intramuscular injection of arginine vasotocin or arginine vasopressin at doses from 0.001 to 0.05 µg/100 g body mass on the background of a water load. Water (5 ml/100 g body mass) was administered through a catheter into stomach to suppress secretion of endogenous antidiuretic hormone (ADH). In experiments with water administration, diuresis increased due to a decrease of osmotic permeability of renal tubules and to excretion of osmotically free water, with the constant clearance of sodium ions. Injection of 0.05 µg arginine vasopressin led to a marked decrease of diuresis due to a rise of reabsorption of osmotically free water without elevation of excretion of osmotically active substances. Injection of the same dose of arginine vasotocin resulted in no increase of diuresis; however, reabsorption of osmotically free water and excretion of osmotically active substances including sodium ions were more pronounced. Hence, both vasotocin and vasopressin increased osmotic permeability of the tubular epithelium, but vasotocin, unlike vasopressin, promoted reduction of reabsorption of sodium ions and their loss with urine. A suggestion is made that one of the reasons for replacement in mammals of the molecular ADH forms (vasotocin by vasopressin) was the absence of the pronounced natriuretic effect in arginine vasopressin. This was of crucial significance to preserve sodium ions in the organism, to maintain water–salt balance in animals adapted to the terrestrial life, and to provide not only osmo-, but also volumoregulation.     

Carp hemoglobin: Functional analysis and thermodynamics of precise oxygen equilibria according to the simple sequential model of Koshland,Némethy and Filmer

Precise oxygen equilibrium curves for carp hemoglobin were determined at 15 °C in bis-Tris buffer, and in phosphate buffer in the presence and absence of P₆-inositol, and at various temperatures in phosphate buffer. Parameters of the Koshland, Némethy and Filmer (1966) (KNF) simple, sequential models (square and tetrahedral) were estimated by non-linear least-square fit of the experimental data to Hill plots. Non-, negative and positive co-operativity can be fitted by the KNF models. Considering equilibrium arguments, $\text{K}{}^2{}_{\mathrm{<mtext>AB</mtext>}}\text{K}{}_{\mathrm{<mtext>BB</mtext>}}$, the KNF parameter governing the co-operativity of the system, predicts a symmetry conserved mode of action in regions of high, positive co-operativity, and a symmetry non-conserved mode of action in regions of low, non- or negative co-operativity. The simple, sequential, square KNF model fits better the Hill plots than does the simple, sequential, tetrahedral KNF model. From the effect of temperature on carp hemoglobin in phosphate buffer, the heats and entropies of the subunit interaction parameter, $\text{K}{}^2{}_{\mathrm{<mtext>AB</mtext>}}\text{K}{}_{\mathrm{<mtext>BB</mtext>}}$, and of the oxygenation parameters, K_BBK_xBK_tAB and $\text{K}{}^{\mathrm{<mtext>3</mtext><mtext>2</mtext>}}{}_{\mathrm{<mtext>BB</mtext>}}\text{K}{}_{\mathrm{<mtext>xB</mtext>}}\text{K}{}_{\mathrm{<mtext>tAB</mtext>}}$, for the square and tetrahedral models, respectively, were calculated and show the square model to account well for previously published data on the carp hemoglobin molecule. This study indicates that the KNF model, in its simplest form, is capable of explaining many of the functional properties of cooperative systems, as opposed to the Monod, Wyman and Changeux (1965) model which seems only to be a special case of the KNF model in regions of high, positive co-operativity.     

Alteration in virulence characteristics of biofilm cells of Pseudomonas aeruginosa in presence of Tamm-Horsfall protein

Summary Tamm-Horsfall glycoprotein is the most abundant protein in human urine. The present investigation was planned to study the effect of Tamm-Horsfall protein (THP) on elaboration of virulence factors by biofilm cells of Pseudomonas aeruginosa. It was observed that with increase in concentration of THP from 10 to 50 μg/ml there was significant enhancement in elaboration of all the virulence factors by biofilm cells of P. aeruginosa. However, with further increase in concentration of THP from 50 to 70 μg/ml, significant decrease in elaboration of all the virulence traits was observed. Implications of these findings in relation to urinary tract infections caused by P. aeruginosa have been discussed.     

Mode of action of sulfoxides in preventing loss of activity of 11β-hydroxylase in cultured bovine adrenocortical cells

Previously, loss of 11β-hydroxylase activity when adrenocortical cells are incubated with the pseudosubstrate cortisol was found to be reduced when the concentration of oxygen was lowered, or when butylated hydroxyanisole (BHA) or dimethyl sulfoxide (Me₂SO) were included in the medium. In the present experiments, we tested the hypothesis that Me₂SO protects 11β-hydroxylase by scavenging OH^? radicals. Substances known to react with OH^? at high rates and non-toxic enough to be used at concentrations of 10–100 mM, including several alcohols, benzoate and radioprotectant thiols, did not prevent loss of activity of 11β-hydroxylase in the presence of 50 μM cortisol. Two of the alcohols, ethanol and glycerol, as well as Me₂SO, were radioprotective in cultured bovine adrenocortical cells. Therefore free OH^? radicals do not appear to be involved in loss of 11β-hydroxylase activity. When sulfoxides other than dimethyl sulfoxide were tested for their ability to protect 11β-hydroxylase in the presence of cortisol, several aryl sulfoxides, particularly dibenzyl sulfoxide, as well as dipropyl sulfoxide, were active at concentrations to $\text{1}\text{200}$ of that required for Me₂SO. Previously, we have demonstrated that 11β-hydroxylase inhibitors, particularly metyrapone, effectively protect against loss of 11β-hydroxylase activity in the presence of pseudosubstrates and therefore we examined whether sulfoxides may act by directly inhibiting 11β-hydroxylase. Me₂SO showed an ED₅₀ for inhibition of 11β-hydroxylase activity of > 1 M, in contrast to its ED₅₀ for protection of 34 mM. For metyrapone, however, the ED₅₀ for inhibition of the enzyme (250 nM) was close to that for protection of activity (270 nM). The other sulfoxides showed ED₅₀-values for inhibition of 11β-hydroxylase that were substantially higher than the ED₅₀-values for protection. Sulfoxides may have a mixed mode of action in protection of 11β-hydroxylase activity, as previously shown for phenols; they may protect by radical scavenging, but may also need to bind close to the active site of the enzyme where destructive radicals may be formed.     

Glycosylation ofα 1 glycoprotein in septic shock: Changes in degree of branching and in expression of sialyl Lewisx groups

The occurrence of differences in acute-phase response, with respect to concentration and glycosylation of ₁-acid glycoprotein (AGP) was studied in the sera of patients surviving or not from septic shock. Crossed affino-immunoelectrophoresis was used with concanavalin A andAleuria aurantia lectin for the detection of the degree of branching and fucosylation, respectively, and the monoclonal CSLEX-1 for the detection of sialyl Lewis^x (SLeX) groups on AGP. Septic shock apparently induced an acute-phase response as indicated by the increased serum levels and changed glycosylation of AGP. In the survivor group a transient increase in diantennary glycan content was accompanied by a gradually increasing fucosylation and SLeX expression, comparable to those observed in the early phase of an acute-inflammatory response. Remarkably, in the non-survivor group a modest increase in diantennary glycan content was accompanied by a strong elevation of the fucosylation of AGP and the expression of SLeX groups on AGP, typical for the late phase of an acute-phase response. Our results suggest that these changes in glycosylation of AGP can have a prognostic value for the outcome of septic shock.Abbreviations AAL Aleuria aurantia lectin - AGP ₁-acid glycoprotein - CAIE crossed affinoimmunoelectrophoresis - Con A Concanavalin A - HSPC human serum protein calibrator - IL-1 interleukin 1 - IL-6 interleukin 6 - LIF leukaemia inhibitory factor - LPS lipopolysaccharide - SLeX sialyl Lewis^x - TNF tumour necrosis factor     

Histoenzymorphology of β-D-glucuronidase in cortical granules in oocytes of some representative vertebrates

Summary Histoenzymorphologic investigations on -D-glucuronidase in cortical granules in oocytes of some representative vertebrates revealed interesting differences in localisation of the enzyme in the two techniques employed using 8-hydroxyquinoline -D-glucosiduronic acid and naphthol AS-BI -D-glucosiduronic acid as substrates. In the former a nonspecific localisation in the entire cytoplasm of the oocytes excepting nucleus was evident whereas in the latter the cortical granules alone exhibited intense activity. To some extent this difference could be accounted for on the basis of yolk content of the eggs. -D-glucoronidase-rich lysosomal cortical granules were found in the amphibian, reptilian and avian oocytes whereas mammalian oocyte cortical granules were devoid of this enzyme. -D-glucuronidase in cortical granules was sensitive to low concentrations of Hg⁺⁺, Cu⁺⁺, glucuronic and mucic acids. Other histochemical evidence for the lysosomal nature of the cortical granules is presented and discussed. -D-glucuronidase seems to have a role in ovum-nutrition and activation which, thus, make a new addition to the existing knowledge of physiological role of -D-glucuronidase and lysosomes.Dr. A. T. Varute — Research Project guide.