Formation of N epsilon-(gamma-glutamyl)-lysine isodipeptide in Chinese-hamster ovary cells.

N epsilon-(gamma-Glutamyl)-lysine isodipeptide was detected in a protein-free fraction of Chinese-hamster ovary cells and their culture fluid by using radioactive lysine as a tracer. The identity of the isodipeptide was established by its separation on ion-exchange chromatography, analysis by h.p.l.c. after derivatization, recovery of lysine after acidic hydrolysis or after cleavage by a specific enzyme, namely gamma-glutamylamine cyclotransferase. The amount of isodipeptide was raised (460 pmol/10(7) cells and 61 pmol/ml of culture fluid were observed as highest values) as the cell density increased. Effects of inhibitors of intracellular protein degradation have shown that the isodipeptide derives from cross-linking N epsilon-(gamma-glutamyl)-lysine bonds formed by tissue transglutaminase. Estimated half-life values of cross-linked proteins were about 3 h. gamma-Glutamylamine cyclotransferase, which may split the isodipeptide formed during the continuous turnover of cross-linked proteins, was also found in Chinese-hamster ovary cells. Isodipeptide may have been accumulated when either its generated amount is beyond the capacity of gamma-glutamylamine cyclotransferase or it is generated in cell compartments where this enzyme is not present.     

Determination of epsilon (gamma-glutamyl)lysine crosslink in proteins using phenylisothiocyanate derivatization and high-pressure liquid chromatographic separation

A sensitive, reproducible high-performance liquid chromatographic method is reported for the determination of the epsilon (gamma-glutamyl)lysine isopeptide bond, which is usually formed by protein-crosslinking transglutaminases between polypeptide chains. The procedure is based on the separation and quantitation of epsilon (gamma-glutamyl)lysine isodipeptide following exhaustive proteolytic digestion of the crosslinked peptide. It involves preliminary separation steps on a cation exchanger resin and a silica HPLC column, precolumn derivatization with phenylisothiocyanate, and reversed-phase high-pressure liquid chromatographic separation on a C18 column. The derivatized isodipeptide gave a linear concentration-response relationship, with a detection limit of 10 pmol/mg of protein. The combination of the preliminary separation steps and the sensitive detection system permits the determination of the epsilon (gamma-glutamyl)lysine crosslink in complex biological systems including total tissue homogenates.     

The end product of transglutaminase crosslinking: simultaneous quantitation of [Nepsilon-(gamma-glutamyl) lysine] and lysine by HPLC-MS3

Transglutaminases catalyze the formation of Nepsilon-(gamma-glutamyl) isodipeptide crosslinks between proteins. These enzymes are thought to participate in a number of diseases, including neurological disease and cancer. A method associating liquid chromatography and multiple stage mass spectrometry has been developed for the simultaneous quantitation of [Nepsilon-(gamma-glutamyl) lysine] isodipeptide and lysine on an ion trap mass spectrometer. Highly specific detection has been achieved in MS3 mode. The method includes a derivatization step consisting of butylation of carboxylic groups and acetylation of amide groups, a liquid-liquid extraction, and a 19-min separation on a 100x2.1-mm Beta-basic C18 column with an acetonitrile gradient elution. 13C6-(15)N2 isotopes of the isodipeptide and the lysine serve as internal standards. The assay was linear in the range of 50 pmol/ml to 75 nmol/ml for the isodipeptide and the range of 10 nmol/ml to 3.5 micromol/ml for the lysine, with correlation coefficients greater than 0.99 for both ions. Intra- and inter-day coefficients of variation ranged from 3.5 to 15.9%. The method was successfully applied to human biological samples known to be crosslinked by transglutaminase such as cornified envelopes of epidermis, fibrin, and normal and Huntington disease brain.     

Epidermal characteristics of reproductive subsets in an inshore population of winter flounder, Pseudopleuronectes americanus

The winter flounder, Pseudopleuronectes americanus (Walbaum), population off Newfoundland can include a 'non-reproductive post-mature' subset at any time together with 'reproductive' and 'immature' subsets. Skin samples were taken from male and female specimens from each subset in the winter, after reproductive flounder have normally undergone a pre-winter phase of gonadal recrudescence, and also during the spring prespawning period and in the summer following spawning. Results for immature flounder demonstrate that fish size influences epidermal thickness in this species. The epidermis of reproductive flounders is thick, representing a secondary sexual characteristic, during gonadal recrudescence. A thick epidermis is most pronounced on the blind-side, particularly in prespawning males. Although the epidermis of non-reproductive post-mature fish was thinner than in prespawning males and females, it was thicker than in immature flounders. Also, the epidermal thickness of non-reproductive post-mature individuals can still display some seasonal change although gonadal recrudescence has not been sustained. There is also a relationship between condition factor and epidermal thickness which is manifested particularly clearly in immature and non-reproductive post-mature flounder.     

Ontogenetic variation in habitat associations for four flatfish species in the Gulf of Maine-Georges Bank region

Multivariate ordination techniques were used to examine how size classes of four flatfish species, American plaice Hippoglossoides platessoides , winter flounder Pseudopleuronectes americanus , yellowtail flounder Limanda ferruginea and fourspot flounder Paralichthys oblongus , are related to bottom depth, bottom temperature, substratum grain size and temporal factors using a 35 year time series from autumn and spring in the Gulf of Maine-Georges Bank region. Depth had the most explanatory value during both seasons in most cases, with fish size generally increasing with depth. One exception was yellowtail flounder in the spring for which a temporal factor explained the most variance, reflecting an increase in size over the time series due to changes in fishing pressure. Temperature was secondarily important for yellowtail flounder in the autumn and for fourspot flounder in both seasons. Substratum type was secondarily important for winter and yellowtail flounders in the spring with smaller fish associated with larger substratum types. Seasonal associations with depth, temperature and substratum are related to seasonal spawning migrations, thermal preferences and other ecological factors.     

Pharmacokinetics of morphine in fish: winter flounder (Pseudopleuronectes americanus) and seawater-acclimated rainbow trout (Oncorhynchus mykiss)

We made a single intraperitoneal (IP) injection of morphine sulfate (40 mg/kg) into winter flounder and seawater acclimated rainbow trout at 10 degrees C and then followed its disposition by measuring the change in plasma morphine concentration for 100 h using a morphine specific ELISA. Disposition also was followed for 6h after a single IV injection of 7.5mg morphine sulfate in winter flounder. Plasma morphine reached a maximum within an hour post-injection IP and then decreased in a bi-exponential fashion with a rapid distribution phase followed by a slower elimination phase. The disposition was slower in flounder than in trout even though the fish were held at the same temperature. For example, plasma clearance was 76 mL h(-)(1) kg(-)(1) in the flounder but was almost twice as much in the trout (153 mL h(-)(1) kg(-)(1)) and mean residence time was 27.9h in the flounder but was 7.0 h in the trout. The present study is the first comprehensive pharmacokinetic analysis for any analgesic in an ectotherm, and our results show that: 1) significant intra-specific variation exists between fishes: and 2) the disposition of morphine in fish is approximately one order of magnitude slower than it is in mammals. These differences may be due in part to mass specific differences in cardiac output.     

A critical period for nutritional control of early gametogenesis in female winter flounder, Pleuronectes americanus (Pisces: Teleostei)

When female winter flounder ( Pleuronectes americanus ) were subjected to periods of satiation feeding alternating with starvation it was found that the first part of their normal six-month feeding period could be associated with subsequent gametogenesis, whereas feeding in the later part of the normal feeding period was not necessary. Conversely if females were not fed during the first part of the normal feeding season they were likely to become non-reproductive, although high condition fish or a non-spawner could become gametogenic in spite of starvation at this time. Feeding restricted to the later part of the normal feeding season was therefore not generally associated with successful gametogenesis for the following year's spawn. When the first part of the feeding period was further subdivided with satiation feeding limited to one or more months within it, most fish became non-reproductive. The exceptions which became reproductive were females which had high post-spawn condition maintained in the month following the immediate spawning period for the individual fish. A nutritionally sensitive period for early gametogenesis in female winter flounder therefore appears to occur in the early part of the normal feeding season, close to the normal spawning period. The experimentally produced non-reproductive females were generally halted in a previtellogenic stage of development for the most advanced oocytes which is consistent with a nutritionally related inhibition of gametogenesis close to the previous spawning period.     

Degradation of cells dying by apoptosis leads to accumulation of epsilon(gamma-glutamyl)lysine isodipeptide in culture fluid and blood.

epsilon(gamma-Glutamyl)lysine isodipeptide, the end-product of proteolytic digestion of proteins cross-linked by transglutaminase, was detected in culture fluid of neonatal rat hepatocytes and plasma of adult rats. The concentration of the isodipeptide was significantly increased in both when high rate of apoptosis with phagocytosis of dying hepatocytes was produced either by epidermal growth factor in the culture or by lead nitrate-induced hyperplasia with subsequent involution in rats. Specific induction of tissue transglutaminase and the consequent formation of highly cross-linked protein envelopes in apoptotic cells have been previously demonstrated by us in both systems.     

On the low viscosity blood of two cold water,marine sculpins

Summary The viscosities of blood from shorthorn sculpin (Myoxocephalus scorpius), longhorn sculpin (Myoxocephalus octodecemspinosus) and winter flounder (Pseudopleuronectes americanus) were compared using a cone-plate viscometer. Both species of sculpin were almost identical with respect to blood and plasma viscosity at the temperatures (0 and 15°C) and shear rates (2.3–90/s) examined. In contrast, the viscosities of winter flounder blood and plasma were considerably greater than those observed in the sculpins. This difference in blood viscosity between the shorthorn sculpin and the winter flounder persisted over the hematocrit range of 0 to 40% red blood cells. The viscosity of the plasma and the interactions between plasma proteins and red blood cells appeared to be the major reasons for the relatively high viscosity of the flounder blood. Although a proportion of the flounder blood viscosity was attributable to fibrinogen, other plasma proteins also appeared to play a significant role. The relatively low blood viscosity of the sculpin species may confer a circulatory advantage during periods of low water temperatures.     

Changes in sperm parameters of sex-reversed female rainbow trout during spawning season in relation to sperm parameters of normal males

The production of all-female populations has important economic benefits in commercial rainbow trout aquaculture. The procedure commonly implemented to produce all-female stocks centers on the sex reversal of rainbow trout females via the administration of androgens in the early developmental stages, followed by the egg fertilization of normal females with semen from sex-reversed females (srf). However, there is no information regarding the quality of semen from srf rainbow trout throughout the spawning season. This information is critical because the quality of srf semen is highly variable. The aim of the study was to determine the changes in the semen parameters of srf rainbow trout throughout the duration of the spawning season. Sperm concentration, sperm motility parameters, and the biochemical parameters of seminal plasma (protein concentration, antitrypsin activity, osmolality, and lactate dehydrogenase activity) from srf were monitored during the spawning season and compared with normal male rainbow trout. The observed values of sperm, protein concentration, antitrypsin activity, osmolality, and lactate dehydrogenase activity of seminal plasma were all higher in comparison with normal males. Semen from srf was therefore characterized by a lower sperm motility during each period of the spawning season, in comparison with normal males, approximately 1.8, 1.5, and 1.7 times, respectively for the beginning, middle, and end of the spawning season. The percentage of sperm motility from srf and normal males were affected by the spawning season in the same way, as the highest values in the middle of the spawning season demonstrate (60% and 91% for srf and normal males, respectively). Spermatozoa of srf are characterized by a lower speed and a more curvilinear trajectory of movement as compared with that of normal males. The patterns of changes during the spawning season in sperm concentration, sperm motility parameters, as well as osmolality, and lactate dehydrogenase activity of the seminal plasma of srf were different in comparison with normal males. Our results could be important for fish breeders in regard to the spawning control of srf rainbow trout, as well as for the development of short- and long-term sperm storage procedures.     

Predator-prey relations between age-1+ summer flounder (Paralichthys dentatus, Linnaeus) and age-0 winter flounder (Pseudopleuronectes americanus, Walbaum): predator diets, prey selection, and effects of sediments and macrophytes

Laboratory experiments and weekly trammel net surveys in the Navesink River, New Jersey (USA) were used to examine the predator-prey interaction between age-1+ summer flounder (Paralichthys dentatus) and age-0 winter flounder (Pseudopleuronectes americanus). Winter flounder (24-67 mm TL) were the dominant piscine prey of summer flounder (n=95, 252-648 mm TL) collected in trammel nets. We observed a temporal shift in summer flounder diets from sand shrimp (Crangon septemspinosa) and winter flounder, dominant during June and early July, to blue crabs (Callinectes sapidus) and other fishes (primarily Atlantic silversides, Menidia menidia and Atlantic menhaden, Brevortia tyrannus) later in the summer. Variations in prey selection appeared to be related to changes in the spatial distribution of predators and spatio-temporal variation in prey availability. In laboratory experiments, summer flounder (271-345 mm total length, TL) preferred demersal winter flounder to a pelagic fish (Atlantic silversides) and a benthic invertebrate (sand shrimp) prey, and the vulnerability of winter flounder increased with increasing prey body size from 20 to 90 mm TL. Experiments testing habitat effects showed that mortality of winter flounder in three different size classes (20-29, 40-49, 60-69 mm TL) was not influenced by sediment grain sizes permitting differential burial of the prey. However, vegetation enhanced survival, with fish suffering lower mortality in eelgrass (Zostera marina, 15+/-0.04%) than in sea lettuce (Ulva lactuca, 38+/-0.04%) or bare sand (70+/-0.07%) when the macrophytes were planted to produce similar leaf surface areas (5000 cm(2) m(-2)). Prey vulnerability appeared to be related to the role of vision in the predator's attack strategy and prey activity levels.     

Seasonal changes in the epidermis of the brown trout Salmo trutta (L.)

A sexual dimorphism was found in the epidermis of hatchery-reared brown trout. The epidermis of both male and female fish undergoes rhythmical changes in thickness during successive spawning cycles. For most of the year the male has a significantly thicker epidermis than the female. During the spawning period (December and January) there is a highly significant drop in the concentration of mucous cells in the epidermis of the male brown trout. These findings are discussed in the light of our knowledge of the skin structure in other salmonid fish and in relation to possible hormonal factors influencing the epidermis of teleosts.     

Renal excretion of chlorphenol red and related organic acids in the intact flounder Pseudopleuronectes americanus

Renal clearance experiments were performed on unanesthetized winter flounder from which bladder urine was collected continuously and caudal vein blood was sampled periodically; renal tissue was also obtained terminally for comparison of test organic acid content in vivo and after incubation in vitro. Urine flow rates and inulin U/P (urine to plasma concentration) ratios were relatively constant for a given fish and averaged 1.0 ml/hr X kg fish and 2.6, respectively. In contrast, U/P ratios for all three test acids cycled from minima of near 100 to maxima of over 1000 roughly every 24 hr when plasma concentrations of unbound acid were below 1 micron; correction of plasma protein binding was required in the case of chlorphenol red, but not PAH or Diodrast. Both in vivo and in vitro the organic acid content of renal tissue was intermediate between plasma and urine concentrations. These results demonstrate that kidneys of intact flounder exhibit the remarkable concentrative capacity for exogenous organic acids previously observed with isolated tubules and suggest that the tubular urine concentration is established in two steps by cell transport first at the peritubular and second at the luminal membrane. The anterior kidney position and the magnitude of maximal PAH and Diodrast clearances, about 1000 ml/hr X kg flounder, are consistent with most of the cardiac output returning to the heart through the renal portal circulation; a regulatory shunt bypassing the peritubular capillaries is proposed to explain cycling of organic acid clearances to minimal values.     

Effect of acute exposure to crude petroleum on some reproductive hormones in salmon and flounder

1. The concentration of total (free + conjugated) androgens in plasma of sexually mature male salmon and flounder was generally lower in oil-exposed fish. 2. Exposure to crude oil inhibited some testicular development of salmon during the final stages of maturation. 3. Oil exposure had no effect on levels of total plasmatic androgens or estradiol in male and female flounder during gonadal recrudescence. 4. Aryl hydrocarbon hydroxylase activity increased in liver and kidney of both species. 5. Sperm, collected from salmon exposed to oil, fertilized eggs from non-exposed females to produce normal alevins.     

Differential amplification of antifreeze protein genes in the pleuronectinae

Summary The organization of antifreeze protein (AFP) genes in the yellowtail flounder was investigated by Southern blotting and the characterization of clones from a genomic library. This flounder, like the closely related winter flounder, has a set of 10–12 linked but irregularly spaced AFP genes. However, it lacks the tandemly amplified set of 20 such genes that are present in the winter flounder. DNA sequence analysis of a tandemly repeated gene from winter flounder showed that it can code for one of the two most abundant AFP components in the serum. Consistent with this higher AFP gene dosage, the peak serum AFP level in midwinter was 9 mg/ml in the winter flounder and only 4 mg/ml in the yellowtail flounder. A recent amplification of the AFP gene in the winter flounder lineage might be responsible for the higher serum AFP levels in this fish. This increase in gene dosage might have helped the winter flounder colonize the ice-laden, shallow-water niche that it currently occupies along the east coast of North America. Genomic Southern blotting of two other righteye flounders, the smooth flounder and the American plaice, illustrates another example of a differential amplification of AFP genes that correlates with a species' exposure to ice.     

Predatory impact of the green crab (Carcinus maenas Linnaeus) on post-settlement winter flounder (Pseudopleuronectes americanus Walbaum) as revealed by immunological dietary analysis

Predation on flatfish during the early juvenile stage is an important factor regulating year-class strength and recruitment. In this study, immunological dietary analysis was performed on green crabs (Carcinus maenas) collected from the Niantic River, Connecticut, in an effort to evaluate the predatory impact of this species on post-settlement winter flounder (Pseudopleuronectes americanus). Through the use of species-specific antiserum, winter flounder proteins were identified in 4.8% of the green crab stomachs analyzed (n = 313, size range = 14-74 mm carapace width, CW), revealing that crabs ≥ 29 mm CW are predators of post-settlement winter flounder in natural populations. The most significant factor underlying the predator-prey interaction was the relative size relationship between species, such that the incidence of winter flounder remains in the stomach contents of green crabs was positively correlated with predator-to-prey size ratio. Results from dietary analysis were incorporated into a deterministic model to estimate the average daily instantaneous mortality and cumulative mortality of winter flounder owing to green crab predation. Accordingly, green crabs may account for 0.4% to 7.7% (mean = 2.2%) of the daily mortality of winter flounder and consume 1.1% to 32.3% (mean = 10.2%) of the flounder year-class. Model simulations further indicate that variations in green crab abundance and size-structure account for the greatest variability in winter flounder mortality. Relative to other macro-crustacean predators, however, predation by green crabs has a minimal effect on winter flounder survival, due in large part to the low densities of these crabs in temperate estuaries.     

Gonadal development and associated changes in liver size and sexual steroids during the reproductive cycle of captive male and female Atlantic cod (Gadus morhua L.)

Gametogenesis in female and male Atlantic cod (Gadus morhua L.) was investigated by sampling blood plasma and gonadal tissue from 19 to 33-month-old fish. The reproductive cycles of both female and male Atlantic cod are characterized by distinct annual variations in gonadal size and developmental stage and these are associated with changes in sex steroids and liver size. I(H) did not change during early gonadal development, but both spent females and males had lower I(H) than late maturing females and spermiating males, respectively. In females I(G) was correlated to plasma E2 levels and they were highest in spawning females. The lowest levels during the reproductive cycle were observed in spent females. Plasma T levels were low throughout ovarian development, and were at a minimum in spent females. 11-ketotestosterone in plasma of males increased rapidly during spermiation, while T increased at earlier testicular stages and reached maximum during spermiation. High plasma levels of steroids in male and female cod during spawning serve to promote further development and growth of less advanced stages of germ cells.     

Immuno-cross reactivity of transglutaminase and cornification marker proteins in the epidermis of vertebrates suggests common processes of soft cornification across species

In differentiating mammalian keratinocytes proteins are linked to the plasma membrane by epidermal transglutaminases through N-epsilon-(gamma-glutamyl)-lysine isopeptide bonds to form the cornified cell envelope. The presence of transglutaminases and their protein substrates in the epidermis of nonmammalian vertebrates is not known. The present study analyses the presence and localization of the above proteins in the epidermis using immuno-cross reactivity across different classes of amniotes. After immunoblotting, some protein bands appear labelled for loricrin, sciellin, and transglutaminase in most species. These proteins are scarce to absent in the epidermis of aquatic species (goldfish and newt) where a stratum corneum is absent or very thin. The molecular weight of transglutaminase immunoreactive bands generally varies between 40 to 62 kDa, with the most represented bands at 52-57 kDa in most species. The more intense loricrin- and sciellin-immunoreactive bands are seen at 50-55-62 kDa, but are weak or absent in aquatic vertebrates. Loricrine-like immunoreactivity is present in the epidermis where alpha-(soft)-keratinization occurs. Isopeptide bonds are mainly associated to bands in the range of 50-62 kDa. In vertebrates where hard-keratin is expressed (the beta-keratin corneous layer of sauropsids and in feathers) or in hair cortex of mammals, no loricrin-like, transglutaminase-, and isopeptide-bond-immunoreactivities are seen. Immunoblotting however shows loricrin-, sciellin-, and trasnsglutaminase-positive bands in the corneous layers containing beta-keratin. Histologically, the epidermis of most amniotes shows variable transglutaminase immunoreactivity, but isopeptide-bond and sciellin immunoreactivities are weak or undetactable in most species. The limitations of immunohistochemical methods are discussed and compared with results from immunoblotting. In reptilian epidermis transglutaminase is mainly localized in 0.15-0.3 microm dense granules or diffuse in transitional alpha-keratogenic cells. In beta-keratogenic cells few small dense granules show a weak immunolabeling. Transglutaminase is present in nuclei of terminal differentiating alpha- and beta-keratinocytes, as in those of mature inner and outer root sheath. The present study suggests that keratinization based on loricrin, sciellin and transglutaminase was probably present in the stratum corneoum of basic amniotes in the Carboniferous. These proteins were mainly maintained in alpha-keratogenic layers of amniotes but decreased in beta-keratogenic layers of sauropsids (reptiles and birds). The study suggests that similar proteins for the formation of the cornified cell envelope are present in alpha-keratinocytes across vertebrates but not in beta-keratinocytes.     

Effects of advanced photoperiod cycle on the epidermis and gonadosomatic index of 2-year-old rainbow trout, Salmo gairdneri R., reared at natural temperature

Sexual dimorphism was shown in the epidermis of 2-year-old rainbow trout. The changes in the epidermis which appear normally at the spawning time can be timed earlier by advancing the normal annual photoperiod cycle by three months.
The epidermis of the advanced fish was significantly thicker than that of the control fish during the winter preceding spawning. For most of the year the epidermis of the males was thicker than that of the females, but females had more mucous cells in the epidermis than males. The maximum concentration of plasma testosterone coincided with the epidermal thickening.     

Reproduction of the summer flounder, Paralichthys dentatus (L.)

The reproduction of summer flounder, Paralichthys dentatus (L.), occurring in the Middle Atlantic Bight was studied from 1974–1979. Males dominated the length interval between 21–35 cm t.l. and were essentially absent in samples >55 cm t.l. Females were more abundant in all length intervals >45 cm t.l. Length at maturity ranged from 24–27 cm t.l. for males and from 30–33 cm t.l. for females which coincided with length at age two. Ovarian egg diamerter frequencies indicated summer flounders are serial spawners and the trend in mean and maximum maturity indexes (% ovary weight of total fish weight) indicated spawning began in September and continued through February in 1975 and probably into March in 1976. Annual condition cycle of males peaked in September and was lowest in April while females' cycle reached a maximum in October which coincided with peak spawning time. Fecundity was related to length, weight and ovary weight for 1974–1977 and the length-fecundity equation F = 0.0007975 L ^3.402 was found to be the best predictive relationship. The overall reproductive strategy of summer flounder was discussed and apparently tends to maximize reproductive potential through an extended spawning season, early maturation, high fecundity, serial spawning and extensive spawning migrations.