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).     

Histology and carbohydrate histochemistry of the alimentary canal in the rainbow trout Oncorhynchus mykiss

A histological and histochemical analyses were carried out on the entire alimentary canal of the rainbow trout Oncorhynchus mykiss . In particular the oesophageal region showed presence of terminal β‐D‐galactose(1–3)‐N‐acetylgalactosamine and α‐N‐acetylgalactosamine. In the anterior and posterior regions of the stomach, lining epithelium and gastric pits exhibited the presence of β‐gal and α‐GalNAc. In addition sialoglycoconjugates having sialic acid–β–galactose(1–3)‐N‐acetylgalactosamine and sialic acid‐N‐acetylgalactosamine as terminal tri‐ and di‐saccharides, were demonstrated. In proximal and distal intestine goblet cells showed the presence of sialoglyconjugates, having sialic acid‐β‐gal(1–3)‐GalNAc and sialic acid‐GalNAc as terminal sequences, belonging to N‐linked chains. In the enterocytes of the entire intestine, terminal GlcNAc, α‐Gal, α‐fucose were found.     

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.     

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.     

Dispersal patterns and status change in a co‐operatively breeding cichlid Neolamprologus pulcher: evidence from microsatellite analyses and behavioural observations

Genetic techniques and long‐term behavioural observations were combined to investigate dispersal patterns and changes in social position in Neolamprologus pulcher , a co‐operatively breeding cichlid from Lake Tanganyika. Comparisons of genetic variance ( F _ST) across sub‐populations demonstrated that fish were genetically more similar to individuals from proximate sub‐populations compared to individuals from distant sub‐populations. Microsatellite analyses revealed year‐long philopatry for some individuals and that other individuals dispersed to new territories and sub‐populations. Individuals appeared to disperse farther (across many territories in a sub‐population or to new sub‐populations) to achieve breeding status. Non‐breeding group members (or helpers) were observed to inherit breeding positions and male breeders were replaced faster than female breeders. These results demonstrate that important and difficult to obtain life‐history information can be obtained from genetic sampling.     

Occurrence of the parasite Glugea hertwigi in young‐of‐the‐year smelt in Lake Tuusulanjärvi

Young‐of‐the‐year smelt Osmerus eperlanus in Lake Tuusulanjärvi were examined for Glugea hertwigi cysts. Cysts were visible on smelt in the beginning of August and showed a peak at the end of August. Glugea hertwigi may cause mortality among the most heavily infected young‐of‐the‐year smelts.     

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.     

Interdemic variation in haematocrit and lactate dehydrogenase in the African cyprinid Barbus neumayeri

This study evaluated whether the African cyprinid Barbus neumayeri from Rwembaita Swamp (low‐oxygen) and Njuguta River (high‐oxygen) in the Kibale National Park, Uganda differed in traits related to aerobic and anaerobic metabolic potential. Haematocrit was measured as an index of blood oxygen‐carrying capacity, and tissue activities and isozyme composition of lactate dehydrogenase (LDH) were measured as indices of tissue anaerobic capacity. To address whether site‐dependent differences were acute responses v . longer‐term adjustments to environmental conditions, these variables were measured in fish sampled shortly after collection and after laboratory maintenance under well‐aerated conditions. In fish sampled in the field, those from the low‐oxygen site had significantly higher haematocrit, but this difference disappeared after long‐term laboratory maintenance. In contrast, fish from the low‐oxygen site had higher liver LDH activities than fish from the high‐oxygen site, and this difference persisted during laboratory maintenance. Polymorphism was detected at both the LDH‐A and LDH‐B loci, and genotype frequencies for LDH‐B differed significantly between collection sites. These results demonstrate physiological, biochemical and genetic differences in B. neumayeri from habitats differing in dissolved oxygen availability and suggest both acute and long‐term responses to local environmental conditions.     

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.     

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.     

Introgressive hybridization between Dolly Varden Salvelinus malma and white‐spotted charr Salvelinus leucomaenis on Hokkaido Island, Japan

Intra‐ and interspecific phylogenetic analysis of Dolly Varden Salvelinus malma and white‐spotted charr Salvelinus leucomaenis throughout Hokkaido Island was conducted using nucleotide sequences of the mitochondrial DNA control region and three nuclear DNA markers [ribosomal DNA (ITS‐1), microsatellite ( u ‐85), SINE ( Fok ‐223)]. A total of 19 mtDNA haplotypes from 271 individuals of Dolly Varden, nine haplotypes from 134 white‐spotted charr were defined, and identified two well‐supported monophyletic clades for each species. Two haplotypes of Dolly Varden, however, were clustered together in the white‐spotted charr clade. Those haplotypes were found only from several rivers in the Shiretoko Peninsula, and a neighbouring river of that region. Analyses of ribosomal DNA and Fok ‐223 loci revealed that both species are characterized by having completely species‐specific diagnostic sequence and fragment patterns. Dolly Varden of the Shiretoko populations are typically allopatric in distribution where white‐spotted charr do not currently occur. In addition, incongruence in genetic relationships between mtDNA and nuclear DNA markers give strong evidence of historical mtDNA introgression between Dolly Varden and white‐spotted charr. Some white‐spotted charr diagnostic alleles in a nuclear microsatellite locus ( u ‐85) were found in some Shiretoko Dolly Varden populations, suggesting that introgressive hybridization might have also occurred in relatively recent contact and potentially ongoing evolutionary event. The present study presents an example of historical hybridization and introgression at the southernmost distribution limits of Dolly Varden.     

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.     

Effects of ethanol preservation on otolith microchemistry

Solution‐based inductively coupled plasma‐mass spectrometry was used to examine the effects of exposure time to ethanol (0, 1, 3, 9, 27 and 81 days) and ethanol quality (ACS‐ v . HPLC‐grade) on strontium (Sr) and barium (Ba) concentrations in sagittal otoliths of hatchery‐raised and wild‐caught young‐of‐the‐year walleye Stizostedion vitreum . No effect of either attribute on Sr and Ba concentrations were detected, indicating that metabolically inert elements that replace calcium in the calcium carbonate matrix ( e.g . Sr and Ba) are not influenced by storage in 95% ethanol.     

Diel activity patterns of juvenile Atlantic salmon in rivers with summer water temperature near the temperature‐dependent suppression of diurnal activity

Direct day and night underwater observations of juvenile Atlantic salmon Salmo salar during summer and autumn showed a duality in response to temperature between 7 and 11° C for young‐of‐the‐year (YOY) Atlantic salmon. They were predominantly diurnal in early summer and nocturnal in late summer although water temperatures were similar. Post‐YOY Atlantic salmon did not show a strong response to temperature as they were mostly nocturnal during the study period. It is suggested that the difference in activity patterns between YOY and post‐YOY Atlantic salmon can be explained by size‐dependent trade‐off between growth and predation risk.     

Carbohydrate levels among winter wheat cultivars varying in freezing tolerance and snow mold resistance during autumn and winter

The LT₅₀ values and soluble carbohydrate levels in wheat ( Triticum aestivum L.) crowns and leaves were monitored throughout autumn and winter in cultivars varying in freezing tolerance and snow mold resistance during 1993/1994 and 1994/1995 in the field at Sapporo, Japan. During the first stage of hardening, from sowing to mid‐November, the pattern of accumulation of mono‐ and disaccharides was similar for all cultivars. During the second stage of cold hardening, from mid‐November to mid‐December, the greatest accumulation of mono‐ and disaccharides, without a corresponding increase in fructan, was observed among the freezing‐tolerant cultivars; and levels of simple saccharides rapidly decreased under snow cover. Conversely, levels of mono‐ and disaccharides in snow mold‐resistant cultivars were less than 70% of those in freezing‐tolerant cultivars before snow cover and maintained low levels throughout winter, while polysaccharide levels in snow mold‐resistant cultivars were about 120% of those in freezing‐tolerant cultivars in December. Sugar metabolism during the winter was examined using 18 cultivars in 1994/1995. LT₅₀ values were correlated to the greatest extent with total mono‐ and disaccharide and fructan content among wheat cultivars excluding snow mold‐resistant cultivars in December. Snow mold‐resistant cultivars tended to metabolize carbohydrates more slowly until the end of the snow cover period. This result suggested that the enzymatic metabolism of the synthesis of sugars and the conversion of fructan to cryoprotective sugars in response to low temperatures, especially subzero ones, might be different between the two contrasting types in resistance to winter stress.     

Dose responses of two pea lines to ultraviolet‐B radiation (280–315 nm)

UV‐B dose responses of two lines of pea were quantified at 2.3, 4.6, 6.9 and 9.2 kJ m⁻² day⁻¹ UV‐B (weighted according to Caldwell's generalised plant action spectrum) in controlled environments providing near‐field doses of photosynthetic radiation. Increasing UV‐B significantly increased UV‐B absorbing compounds in both lines. In the UV‐B sensitive line, JI1389, increasing UV‐B significantly inhibited most aspects of plant morphology and biomass. In the more UV‐B‐tolerant line, Scout, increasing UV‐B significantly reduced foliage area but had no effect on above‐ground biomass, although root biomass was significantly increased. Reduced plant height in JI1389 was caused by shorter internodes, in turn due to reduced cell number but not cell length. UV‐B had no significant effects on photosynthesis in either line. Significant dose responses were linear for the growth of the main stem in JI1389 but remaining significant dose responses were better fitted by quadratics with maximum UV‐B effects occurring in the range 5–7 kJ m⁻² day⁻¹ PAS300, due to stimulation of branch growth at the highest dose. However, growth stimulation by UV‐B was confined to PAS300 doses which at temperate latitudes would result only from rather extreme ozone depletions. We conclude that investigations using relatively low UV‐B doses, rather than those well above the current maximum, may be the best approach to both understanding of the fundamental mechanisms of plant responses to UV‐B and quantifying the magnitude of responses to stratospheric ozone depletion.     

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.     

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.     

Scavenging of superoxide and hydrogen peroxide in blue‐green algae (cyanobacteria)

Blue‐green algae (cyanobacteria) have evolved as the most primitive, oxygenic, plant‐type photosynthetic organisms. Within a single prokaryotic cell, they have uniquely accommodated both oxygenic photosynthesis and aerobic respiration, which are known to produce superoxide and hydrogen peroxide as inevitable byproducts. Two types of superoxide dismutase have been characterized in both N₂‐fixing and non‐N₂‐fixing cyanobacteria, namely cytosolic iron‐containing superoxide dismutase and thylakoid‐bound manganese‐containing superoxide dismutase. No qualitative differences between various cell types (vegetative cells, heterocysts) were found. In contrast to chloroplasts, most of the cyanobacterial species show catalatic activity. From two species the corresponding enzymes have been characterized as typical prokaryotic (bifunctional) catalase‐peroxidases with homologies to cytochrome c peroxidases and ascorbate peroxidases. In addition to catalatic activity, some strains exhibit ascorbate peroxidase activity, but to date there are no reports detailing purification and characterization.
Cyanobacteria were found to contain low intracellular ascorbate concentrations (30‐100 µ M ) and 2‐5 m M glutathione. Both monodehydroascorbate and glutathione reductase activities were detected in most species examined, whereas dehydroascorbate reductase activity was absent. The question as to whether a glutathione‐ascorbate cycle exists in cyanobacteria cannot be answered at present.