Air‐breathing Behavior of the Jeju Fish Hoplerythrinus unitaeniatus in Amazonian Streams

The breathing behavior of the jeju fish Hoplerythrinus unitaeniatus has been the focus of several studies in recent decades. Few of these studies, however, have described how the fish's air breathing functions in natural environments. We examined changes in the behavior of H. unitaeniatus during daily variations in the dissolved‐oxygen (DO) content of the water in Amazonian streams. We recorded the mean time intervals between instances when the fish breached the water surface to take in atmospheric air and the variation in the DO concentrations. We also observed the number of individual fish that breached the surface together as well as their territory‐forming behavior. We recorded the individual and collective anti‐predator strategies in the presence of a potential predator, experimentally testing the capacity of H. unitaeniatus to recognize the shape of a predator. The results indicated that as the DO concentration decreased, the individuals of H. unitaeniatus tended to breach the water surface at shorter time intervals, which increased their exposure to predators. To minimize the risks from this longer exposure time, the species tends to adopt anti‐predator strategies individually or as a group. In well‐lit locations, the fish recognized the predator's silhouette, left their territory and took a longer time to return. We provide an original contribution to the understanding of the anti‐predatory tactics of the species by describing the behavior of group formation and territory definition as a tool to maximize efficiency in the defense against predators, a behavior that until now was an unknown strategy for this species.     

Postharvest changes in endogenous cytokinins and cytokinin efficacy in potato tubers in relation to bud endodormancy

Using an indirect enzyme‐linked immunosorbent assay (ELISA), the effects of postharvest storage duration and temperature on endogenous cytokinins in potato ( Solanum tuberosum L. cv. Russet Burbank) tuber apical bud tissues in relation to endodormancy status were determined. Following fractionation by HPLC, a total of eight cytokinins were detected and these were: zeatin riboside‐5'‐monophosphate (ZRMP), zeatin‐ O ‐glucoside (ZOG), zeatin (Z), zeatin riboside (ZR), isopentenyl adenosine‐5'‐monophosphate (IPMP), isopentenyl adenine‐9‐glucoside (IP‐9‐G), isopentenyl adenine (IP) and isopentenyl adenosine (IPA). Regardless of postharvest storage temperature or endodormancy status, IP‐9‐G was the most abundant cytokinin detected while ZRMP and ZOG were the least abundant ones. In tubers preincubated at a growth‐permissive temperature (20°C) prior to extraction, the loss of endodormancy was preceded by significant increases in the endogenous levels of Z, ZR, IPMP and IP‐9‐G. When stored continuously at a growth‐inhibiting temperature (3°C), significant increases in ZR, IP‐9‐G and IP + IPA were observed. The total content of cytokinins increased by over 7‐fold during postharvest storage and this increase was a result of de novo biosynthesis. Dose‐response studies using IPA and ZR demonstrated a time‐dependent increase in apparent cytokinin sensitivity during postharvest storage. With the exception of IP‐9‐G, injection of any of these endogenous cytokinins resulted in the rapid and complete termination of tuber endodormancy. The significance of these results with respect to endodormancy regulation and the possible mechanisms controlling cytokinin levels in potato tubers are discussed.     

Glycoconjugate distribution in gastric fundic mucosa of Umbrina cirrosa L. revealed by lectin histochemistry

The stomach of adult shi drum Umbrina cirrosa was investigated using a battery of nine horseradish peroxidase‐conjugated lectins combined with enzymatic treatment, in order to distinguish glycoconjugate sugar residues. Epithelial cells showed the presence of galactosyl(β1→4)N‐acetylglucosamine, mannose, N‐acetylgalactosamine, N‐acetylglucosamine, fucose and sialic acid‐galactosyl(β1→3)N‐acetylgalactosamine residues. Gastric pits had similar sugar residues with the exception of N‐acetylgalactosamine which was less diffused. Gastric glands were characterized by the presence of glycoconjugates containing galactosyl(β1→3)N‐acetylgalactosamine, N‐acetylglucosamine, galactosyl(β1→4) N‐acetylglucosamine, N‐acetylgalactosamine and a small amount of sialic acid linked to N‐acetylgalactosamine.     

Release of sex steroids into the water by roach

Electro‐olfactogram (EOG) recordings of the olfactory epithelium of both male and female roach Rutilus rutilus demonstrated that both sexes were able to detect free and glucuronidated 17,20β‐dihydroxy‐4‐pregnen‐3‐one (17,20β‐P) with high sensitivity. Male, but not female, roach were also sensitive to androstenedione. Sexually mature female roach were shown to release free 17,20β‐P, glucuronidated 17,20β‐P and androstenedione into the water; for all three steroids, the rate of release was significantly enhanced by injection of carp pituitary extract (CPE). A series of trials was also carried out which showed that mature males, and to a lesser extent immature males and females, were able also to release free and glucuronidated 17,20β‐P, both before and after CPE treatment. Water extracts from containers that had held CPE‐treated mature male and female roach were examined for the presence of other steroids. This revealed that free and glucuronidated 17,20β‐P plus free and glucuronidated 17,20β,21‐trihydroxy‐4‐pregnen‐3‐one (17,20β, 21‐P) predominated in water extracts from both sexes. The free moieties of 17,20α‐dihydroxy‐4‐pregnen‐3‐one, 17‐hydroxyprogesterone and 11‐deoxycortisol were found at concentrations which were between four and 20 times lower than those of free 17,20β‐P. Androstenedione was found at concentrations which were 25‐fold lower than those of 17,20β‐P. Despite its apparent high rate of release by sexually mature male and female roach, free 17,20β,21‐P was found not to exhibit any EOG activity at the highest dose tested (10⁻⁷ M).     

Effect of solar radiation (UV and visible) at high altitude on CAM‐cycling and phenolic compound biosynthesis in Sedum album

The field experiment was carried out in order to compare the response of a CAM plant, Sedum album L., to solar radiation at a high altitude (2 100 m) with that at a low altitude location with respect to CAM and phenolic content. Treatment sites included (1) sun‐exposed, low altitude, (2) sun‐exposed, high altitude with different light treatments, including UV‐B and UV‐B + A screening, and (3) shade at high altitude. After a 70‐day treatment period, CAM‐cycling and phenolic compound content were analysed, and high altitude treatments were compared to the low altitude control. The sun‐exposed low altitude control was characterized by CAM‐cycling and a low phenolic compound content during the experiment. In plants transplanted to the high altitude, only the shaded group maintained a CAM‐cycling and a phenolic compound content similar to those of the sun‐exposed low altitude control. Samples under UV‐B and UV‐B + A filters showed similar responses, suggesting the absence of a specific UV‐A radiation effect. The screening of UV‐B or UV‐B + A radiation allowed plants to partially maintain a CAM‐cycling and induced a decrease in phenolic compound content. These responses under UV filters were, however, intermediate between those observed in sun‐exposed and shaded groups. These results demonstrate a specific effect of radiation from both visible (400–800 nm) and UV‐B (280–320 nm) bands on both CAM‐cycling and phenolic biosynthesis in S. album L. plants. These light‐dependent effects are discussed on a physiological basis and a possible interaction between CAM‐cycling and phenolic metabolism is suggested.     

Peroxisomal manganese superoxide dismutase: Purification and properties of the isozyme from pea leaves

The peroxisomal manganese superoxide dismutase (perMn‐SOD; EC 1.15.1.1) was purified to homogeneity for the first time from peroxisomes of pea ( Pisum sativum L.) leaves. Peroxisomes were isolated from pea leaves by sucrose density‐gradient centrifugation, and then perMn‐SOD was purified from these organelles by two purification steps involving anion‐exchange and gel‐filtration fast protein liquid chromatography. Pure peroxisomal Mn‐SOD had a specific activity of 2 880 units per mg protein and was purified 3 000‐fold, with a yield of about 7 µg enzyme per kg pea leaves. The relative molecular mass determined for perMn‐SOD was 92 000, and it was composed of four equal subunits of 27 kDa. Ultraviolet and visible absorption spectra of the enzyme showed two absorption maxima at 278 and 483 nm, respectively, and two shoulders at 290 and 542 nm. By isoelectric focusing (pH 5‐7), an isoelectric point of 5.53 was determined for perMn‐SOD. In immunoblot assays, purified Mn‐SOD was recognized by a polyclonal antibody against mitochondrial Mn‐SOD (mitMn‐SOD) from pea leaves. The amino acid sequence of the N‐terminal region of the purified peroxisomal enzyme was determined. A 100% identity was found with the mitMn‐SOD from pea leaves, and high identities were also found with Mn‐SODs from other plant species.     

Increased expression of two cDNAs encoding metallothionein‐like proteins during growth of Cicer arietinum epicotyls

The present study was undertaken to identify and characterize clones whose expression increase during Cicer arietinum epicotyl growth. Two cDNAs encoding two different plant metallothionein (MT)‐like proteins have been isolated from a cDNA library from epicotyls of Cicer arietinum L. cv. Castellana. The CanMT‐1 deduced protein appears to have the typical structure of type 1 MT where all Cys residues are in Cys‐X‐Cys motifs, while the CanMT‐2 has the typical structure of type 2 MT having Cys‐Cys and Cys‐X‐X‐Cys motifs within the N‐terminal domain. Both chickpea CanMTs are up‐regulated during epicotyl growth, showing increased expression in mature tissues, mostly CanMT‐1, which is undetectable in young epicotyls. Accordingly, stem of chickpea plants displayed the highest level of CanMT‐1 expression in the basal internode, with reduced growth, decreasing towards the apex. Osmotic stress by PEG, which inhibited growth, and ABA treatment induced the expression of MT‐like genes, which points to a relationship between chickpea MTs and ABA‐mediated stress response. Unlike CanMT‐2, CanMT‐1 is induced in chickpea epicotyls by cadmium indicating a different function for both clones. We conclude that these MT‐like proteins, in particular CanMT‐1, are regulated by the developmental stage and may participate in cell maturation process.     

Population structure of lake‐type and river‐type sockeye salmon in transboundary rivers of northern British Columbia

The population structure of 'lake‐type' and 'river‐type' sockeye salmon Oncorhynchus nerka , primarily in transboundary rivers in northern British Columbia, was examined with a survey of microsatellite variation. Variation at 14 microsatellite loci was surveyed from c . 3000 lake‐type and 3200 river‐type sockeye salmon from 47 populations in six river drainages in British Columbia. The mean F _ST for the 14 microsatellite loci and 47 populations was 0·068, and 0·034 over all river‐type populations. River‐type sockeye salmon were more genetically diverse than lake‐type sockeye salmon, with expected heterozygosity of river‐type sockeye salmon 0·72 and with an average 12·7 alleles observed per locus, whereas expected heterozygosity of lake‐type sockeye salmon was 0·65 with and average 10·5 alleles observed per locus. River drainage of origin was a significant unit of population structure. There was clear evidence of genetic differentiation among river‐type populations of sockeye salmon from different drainages over a broad geographic range in British Columbia.     

Survival and behaviour of post‐smolt Atlantic salmon in coastal habitat with extreme tides

The early marine migration of 55 Atlantic salmon post‐smolts tagged with acoustic transmitters was automatically monitored using 13 to 25 km long arrays of receivers deployed inside the Bay of Fundy, a coastal system on the east coast of Canada. The survival of post‐smolts from groups with short‐ and long‐term transmitters in coastal habitat up to 10 km from the river was 92 to 100%, indicating a successful transition to salt water and departure. Migration for 68 to 77% of post‐smolts followed a direct route and it was rapid (transit time usually <12 h). Post‐smolts initially migrated in a south to south‐west direction ( i.e . orientation towards the mouth of the bay) and they were aggregated near the coast. Post‐smolts with long‐term transmitters were monitored 20 km from the river where they continued to be aggregated, moving near the coast through a 'common corridor', and their survival to that point was at least 84%. Post‐smolts from both groups travelled heading out of the coastal system during ebb tides. Flood tides interrupted migration, and they caused changes in travel direction and delays in departure for post‐smolts not leaving by a direct route. Monitoring of coastal habitat inside the Bay of Fundy intercepted 62% of migrating post‐smolts with long‐term transmitters returning after an initial absence of 2 to 22 days. Returning post‐smolts displayed a resident behaviour, using the habitat monitored inside the Bay of Fundy during July and August.     

Isolation, characterization and significance of papaya β‐galactanases to cell wall modification and fruit softening during ripening

β‐Galactosidases (EC 3.2.1.23) from ripe papaya ( Carica papaya L. cv. Eksotika) fruits having galactanase activities were fractionated by a combination of cation exchange and gel‐filtration chromatography into three isoforms, viz., β‐galactosidase I, II and III. The native proteins of the respective isoforms have apparent molecular masses of 67, 67 and 55 kDa, each showing one predominant polypeptide upon SDS‐PAGE of about 31 and 33 kDa for β‐galactosidases I and III, respectively, and of 67 kDa for β‐galactosidase II. The β‐galactosidase I protein, which was undetectable in immature fruits, appeared to be specifically accumulated during ripening. The β‐galactosidase II protein was present in developing fruits, but its level seemed to decrease with ripening. β‐Galactosidase I seemed to be an important softening enzyme; its activity increased dramatically (4‐ to 8‐fold) to a peak early during ripening and correlated closely with differential softening as related to position in the fruit tissue. The inner mesocarp tissue was softer, and its wall pectins were modified earlier and firmness decreased more rapidly during ripening compared to the outer mesocarp tissue. β‐Galactosidase II also may contribute significantly to softening because of its ability to catalyse increased solubility and depolymerization of pectins as well as through its ability to modify the alkali‐soluble hemicellulose fraction of the cell wall. The physiological significance of both β‐galactosidase isoforms may partly be attributed to their functional capacity as β‐(1,4)‐galactanases.     

Triplophysa rosa sp. nov.: a new blind loach from China

A new blind loach of Triplophysa Rendahl 1933 was collected from a subterranean pool in a cave at Wulong County, Chongqing City, China, in November 2002. The new species, named Triplophysa rosa sp. nov., can be distinguished from its congeners by the following unique characters: eyes vestigial; 9 branched dorsal‐fin rays; 12 branched pectoral‐fin rays; 7 branched pelvic‐fin rays; 6 branched anal‐fin rays; 7 + 7 branched caudal‐fin rays; distal margin of dorsal‐fin concave; tip of pelvic‐fin surpasses vertical level of anus; caudal‐fin deeply forked; whole body scaleless and colourless.     

Facilitated diffusion drives transport of oxidised ascorbate molecules into purified plasma membrane vesicles of Phaseolus vulgaris

Recently, the presence of a carrier‐mediated transport system for ascorbate was demonstrated in the plant plasma membrane. To investigate the possible physiological importance of this system in apoplastic ascorbate metabolism we further characterized this carrier. Transport of Asc was measured by incubating freshly‐purified plasma membrane vesicles from hypocotylar hooks of Phaseolus vulgaris together with [¹⁴C]‐labelled Asc. In this paper we show that ascorbate transport is detectable over a relatively broad pH range (6 to 7.5) and is not affected by protonophore addition. [¹⁴C]‐Ascorbate is not taken up into vesicle fractions consisting of sealed inside‐out oriented vesicles, suggesting that it is transported only from the apoplast to the cytoplasm. Asc uptake into vesicles previously loaded with ascorbate was also tested. Surprisingly, uptake of radioactive molecules was up to 3‐fold higher in the ascorbate‐loaded vesicles compared to non‐loaded control vesicles ( P < 0.001). The uptake of [¹⁴C]‐ascorbate in both the ascorbate‐loaded as the non‐loaded membrane vesicles was inhibited by addition of DTT and not by glutathione or ferricyanide. Based on various observations such as cis ‐inhibition, trans ‐stimulation and insensitivity towards proton gradients, a facilitated uptake mechanism is suggested. Our results strongly indicate that dehydroascorbate is the preferred transported species from the apoplastic to the cytoplasmic side of the membrane. This transport system is possibly involved in the regeneration of apoplastic ascorbate.     

Immunohistochemical study of the gastrointestinal endocrine cells in the Korean aucha perch

The regional distribution and relative frequency of neurohormonal peptides‐producing cells were demonstrated in the gastrointestinal (GI) tract of the Korean aucha perch Coreoperca herzi , using 10 types of specific antisera raised against mammalian regulatory peptides. The GI tract was divided into four portions: stomach, gastro‐intestinal junction, and small and large intestine. Most of the immunoreactive (IR) cells were in the mucosal epithelium and they were generally spindle shaped with a long cytoplasmic process. In addition, ovoid cells were found in the gastric regions. Serotonin‐, somatostatin‐, glucagon‐, cholecystokinin‐8 (CCK‐8)‐ and pancreatic polypeptide (PP)‐IR cells were observed with various relative frequencies. No chromogranin A‐, secretin‐, vasoactive intestinal peptide‐, substance P‐ or bombesin‐IR cells, however, were found. Serotonin‐IR cells occurred throughout the GI tract and were the most numerous. Somatostatin‐IR cells were restricted to the stomach and gastro‐intestinal junction in numerous and moderate frequencies, respectively, but small numbers of glucagon‐IR cells were restricted to the small intestine. Numerous CCK‐8‐IR cells were found in the small intestine but variable numbers of PP‐IR cells occurred throughout the GI tract except for the large intestine. In general the distribution and relative frequency of these IR cells correspond well to previous reports in teleosts but there are some difference in this species.     

c‐erbB‐2 and p53 expression in breast cancer fine needle aspirates

The aim of this study was to co‐evaluate c‐ erbB ‐2 and p53 protein expression in breast cancer fine needle aspirates (FNA) and to compare this with histological variables and the immunohistochemical phenotype of the tumours. Furthermore, we assessed the relationship of c‐ erbB ‐2 and p53 immunocytochemical expression to tumour prognostic factors. We examined 124 breast cancer FNAs and 79 matched surgical specimens using the avidin–biotin complex (ABC) and the alkaline phosphatase immunocytochemical techniques. C‐ erbB ‐2 immunopositivity was detected in 37.9% of the FNAs, while 31.7% were positive for p53. A statistically significant correlation was observed between p53 negativity and absence of c‐ erbB ‐2 immunostaining in the FNAs ( P =0.0007). Smears from infiltrating ductal carcinomas tended to be more frequently positive for p53 (36.7%) than those from lobular carcinomas (11.7%) ( P =0.054). In matched tumour tissues, c‐ erbB ‐2 was positive in 16.7% and p53 in 19% of cases. The immunocytochemical results for both c‐ erbB ‐2 and p53 were significantly correlated with the immunohistochemical results. There was no correlation between c‐ erbB ‐2 and p53 immunostaining, in both FNAs and tissues, and patients' menopausal status, tumour size, grade and lymph node status.     

Swim or hide: predator cues cause species specific reactions in young fish larvae

The anti‐predator behaviour of first‐feeding (9 mm total length) hatchery‐reared pike Esox lucius larvae and wild‐caught three‐spined stickleback Gasterosteus aculeatus larvae was studied in the presence of chemical and visual signals from a fish predator. The results clearly showed that both fish species detected the predator by chemical signals alone but the combined chemical and visual signals caused stronger and more diverse reactions. Subsequent to predator detection, their swimming activity decreased, they attacked zooplankton less frequently and spent more time in the vegetation. Fishes differed in their anti‐predator responses. Pike reacted more clearly to chemical signals alone while three‐spined stickleback needed both chemical and visual cues to assess the predation risk. The strongest reaction was the reduction in swimming activity in the three‐spined stickleback (38% decrease) and a decrease in attack rate of the pike (39% decrease), but only when a refuge was available. Pike were more dependent on the vegetation cover showing almost no anti‐predator responses in the absence of a refuge. In addition, there was a difference in the refuge use of three‐spined stickleback between different macrophytes, indicating a complex or dense structure, which was difficult to penetrate or chemical excretion in one of them.     

Action potentials and variation potentials in sunflower: An analysis of their relationships and distinguishing characteristics

Sunflower plants ( Helianthus annuus L.) were given an electrical stimulus to the stem or a heat (flame)‐wound to a single leaf or a cotyledon. The resulting electrical activity was monitored with extracellular electrodes. An electrical stimulus applied to the stem frequently evoked an action potential (AP), but never a variation potential (VP). In contrast, a heat‐wound applied to a leaf virtually always elicited a VP, which was often accompanied by one or more superimposed spikes (putative APs). The kinetic parameters of the AP and the VP were investigated. The AP appears to propagate without decrement in velocity or magnitude, whereas the VP parameters decrease significantly with distance. The heat stimulus triggered rapid alterations in stem elongation/contraction, which preceded changes in electrical potential, indicating the transmission of a hydraulic signal. Light‐off and light‐on stimuli evoked negative‐ and positive‐going changes in extracellular electrical potential, respectively, corresponding to de‐ and hyper‐polarization of the plasma membrane. Membrane depolarization (extracellularly manifested as a VP) evoked by both the light‐off and heat‐wounding stimuli was able to trigger one or more APs. We interpret these results to suggest that APs are "genuine" electrical signals involving voltage‐gated ion channels or pumps, which can be evoked directly by electrical stimulation or indirectly by changes in membrane potential occurring during the VP or after the light‐off stimulus. In contrast, VPs appear to be a local (non‐transmissible) electrical consequence of the passage of a rapidly transmitted hydraulic signal in the xylem, presumably acting on mechanosensitive ion channels or pumps in adjacent living cells.     

H+/PPi stoichiometry of a membrane‐bound pyrophosphatase of plant mitochondria

The H⁺/PP_i stoichiometry of the mitochondrial H⁺‐PP_iase from pea ( Pisum sativum L.) stem was determined by two kinetic approaches, and compared with the H⁺/substrate stoichiometries of the mitochondrial H⁺‐ATPase, and the vacuolar H⁺‐PP_iase and H⁺‐ATPase. Using sub‐mitochondrial particles or preparations enriched in vacuolar membranes, the rates of substrate‐dependent H⁺‐transport were evaluated: by a mathematical model, describing the time‐course of H⁺‐gradient (ΔpH) formation; or by determining the rate of H⁺‐leakage following H⁺‐pumping inhibition by EDTA at the steady‐state ΔpH. When the H⁺‐transport rates were divided by those of PP_i or ATP hydrolysis, measured under identical conditions, apparent stoichiometries of ca 2 were determined for the mitochondrial H⁺‐PP_iase and H⁺‐ATPase, and for the vacuolar H⁺‐ATPase. The stoichiometry of the vacuolar H⁺‐PP_iase was found to be ca 1. From these results, it is suggested that the mitochondrial H⁺‐PP_iase may, in theory, function as a primary H⁺‐pump poised towards synthesis of PP_i and, therefore, acting in parallel with the main H⁺‐ATPase.     

A Nicotiana plumbaginifolia protein labeled with an azido cytokinin agonist is a glutathione S‐transferase

The mechanisms of reception/transduction of cytokinins still remain largely unknown. We used 1‐(2‐azido‐6‐chloropyrid‐4‐yl)‐3‐(4‐[³H])phenylurea ([³H]azido‐CPPU), a new photoaffinity probe to search for cytokinin‐binding proteins. A soluble protein that binds phenylurea‐type cytokinins has been specifically photolabeled in Nicotiana plumbaginifolia (cv. Viviani line pbH1D) leaf extracts. The protein was purified to homogeneity by affinity chromatography. Its N‐terminal amino acid sequence, as well as four internal peptidic sequences are highly homologous with the theta class of the glutathione S‐transferase superfamily (GST, EC 2.5.1.18) including Hyoscyamus muticus and Arabidopsis GSTs identified as auxin‐binding proteins. The purified N. plumbaginifolia protein also possesses GST enzymatic activity. To test the possible involvement of this GST in the mechanism of action of cytokinin, we studied the binding of tritiated‐CPPU to the purified GST in the presence of various compounds, cytokinin agonists, cytokinin antagonists, or inactive molecules. Thidiazuron is a poor competitor, and neither zeatin nor the active optical isomer R‐MeBA is able to inhibit the binding of CPPU. There is no correlation between the cytokinin activity and the binding properties of the molecules tested. Our results confirmed that plant GSTs bind different compounds, especially plant hormones but probably have no specific role in the mode of action of cytokinins.