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.     

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).     

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.     

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.     

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.     

Cell death caused by a combination of aluminum and iron in cultured tobacco cells

The inhibition of growth of tobacco cells ( Nicotiana tabacum L. cv. Samsun) after treatment with A1 in medium containing high concentrations of cations requires the presence of Fe (II or III) during the treatment. We examined whether the inhibition of the post‐treatment growth is due to cell death occurring during the treatment with A1 and Fe. In cells at the end of A1 treatment, the integrity of the plasma membrane and the integrity of the mitochondrial inner membrane were monitored by use of Evans blue staining and the cleavage of 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyl tetrazolium bromide (MTT), respectively. Time‐course and dose‐response experiments indicate that the inhibition of post‐treatment growth is strongly related to Evans blue uptake, but not to MTT cleavage. These results suggest that the loss of integrity of the plasma membrane caused by a combination of Al and Fe directly contributes to cell death and the inhibition of post‐treatment growth.     

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.     

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.     

Quantitative and qualitative differences in C6‐volatile production from the lipoxygenase pathway in an alcohol dehydrogenase mutant of Arabidopsis thaliana

Six‐carbon (C₆) volatile products are released from the enzymatic action of hydroperoxide lyase (HPL), a component of the lipoxygenase (LOX) pathway and form the basis of the "green‐note" flavour characteristic of many consumed plant products. Arabidopsis leaf tissue contains the C₆‐aldehydes hexanal, and trans ‐2‐hexenal as well as the C₆‐alcohols: hexanol, and 3‐hexenol. Interconversion between C₆‐aldehydes and alcohols is thought to proceed through the action of alcohol dehydrogenase (ADH). Using an ADH mutant of Arabidopsis , we have shown that there are large quantitative and qualitative differences in the accumulation of C₆‐volatiles in the absence of ADH activity. The total quantity of LOX‐derived volatiles is greater on a fresh weight basis in the ADH mutant. Qualitatively, hexanol and 3‐hexenol levels are approximately 62% and 51% lower in the mutant, respectively, whereas levels of hexenal are approximately 10‐fold higher. Hexanal accumulation, however, is unaffected in the mutant. The altered profile of LOX‐derived volatiles does not have an effect on the steady‐state levels of mRNA for allene oxide synthase (AOS) or LOX. HPL activity and mRNA quantity, however, are higher in the mutant relative to wild type, suggesting that altered product levels in the mutant affect HPL regulation.     

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.     

Changes in reproductive physiology and behaviour over the nesting cycle in male three‐spined sticklebacks

Plasma 11‐Ketotestosterone (11 KT) and testosterone (T) levels and spiggin‐mRNA levels, as well as the kidney‐somatic index ( I _K) were measured in sexual males and in paternal males at the middle (5 days paternal) and at the end (8 days paternal with hatched eggs) of the nesting cycle in three‐spined sticklebacks Gasterosteus aculeatus from two populations. Glueing (using threads of 11 KT induced kidney‐protein spiggin) and fanning behaviour was measured daily. Fanning increased in paternal fish and remained low in sexual males. Plasma 11 KT and T levels, on the other hand, declined significantly in parental compared to sexual males as did spiggin expression, I _K and glueing behaviour. Thus, the drastic decrease in circulating 11 KT levels during the later parental phase may have resulted in an energy‐saving decrease in spiggin‐production and glueing, when this was no longer needed for nest maintenance. In addition, the mRNA levels of the β‐subunits of both gonadotropins, luteinising hormone (LH) and follicle stimulating hormone (FSH) were measured. The expression of both gonadotropins declined in the parental phase (not significant for β‐FSH in one of the populations) which was consistent with a decline in androgen levels possibly controlled via decreased gonadotropin secretion.     

Inactivation by gene disruption of 2‐cysteine‐peroxiredoxin in Synechocystis sp. PCC 6803 leads to increased stress sensitivity

2‐Cysteine‐peroxiredoxins (2‐CP) constitute a ubiquitous group of enzymes which reduce toxic alkyl hydroperoxides. In higher plants it was shown that the nuclear encoded 2‐CPs are posttranslationally imported into the chloroplasts, the site of most active oxidative metabolism in plants (Baier and Dietz 1997, Plant J. 12; 179‐190). The genome of the bluegreen alga Synechocystis (EMBL acc. # D64000) encodes a 2‐CP which shares 60% homology to higher plant 2‐CPs on the gene level and about 70% on the level of the mature protein. In order to elucidate the physiological significance of 2‐CPs for photosynthetic organisms, the 2‐CP gene was mutated in Synechocystis sp. PCC 6803 by insertion of a kanamycin gene cartridge. Following complete segregation mutant lines were analyzed for growth and photosynthetic parameters. The mutants revealed decreased growth rates as compared to the wild type. Growth inhibition was relieved after lowering the concentration of Fe or trace elements in the growth medium. Chlorophyll a fluorescence transients as induced by saturating light pulses were used as indicator for the state of photosynthesis. The effective quantum yield decreased at lower light intensities in the mutants as compared to the wild type Synechocystis . Simultaneously, electron transport rates saturated at lower light intensities in the mutants. These data provide the first evidence that 2‐CPs play a pivotal protective role in photosynthesis.     

A new technique to gather 3‐D spatial information using a single camera

A new technique using a single camera and shadows to determine 3‐D spatial positions of fishes in the laboratory is described. The apparatus consisted of a large aquarium (2·0 × 1·5 × 0·4 m), a wide‐angle camera mounted above and two light sources to cast shadows to either side of the fish. Using image analysis and vector mathematics, aquarium objects were plotted within 1·5 cm of their actual location along the x ‐, y ‐ and z ‐axis. The technique was also successful in quantifying changes in 3‐D spatial pattern of juvenile fish, Atlantic cod Gadus morhua (7·4–8·6 cm standard length, L _S) and cohabitant piscivorous shorthorned sculpin Myoxocephalus scorpinus (12·0–25·8 cm L _S), at these same viewing fields. The new technique should have a wide application, largely because it is potentially less expensive, laborious and invasive than alternative methods for determining 3‐D positions of fishes.     

Abscisic acid involvement in the induction of summer‐dormancy in Poa bulbosa, a grass geophyte

Summer‐dormancy occurs in geophytes that inhabit regions with a Mediterranean climate (mild, rainy winters and hot, dry summers). The environmental control of summer‐dormancy and the involvement of phytohormones in its induction have been little studied. Poa bulbosa L. is a perennial grass geophyte in which summer‐dormancy is induced by long days and by high temperature. Prolonged treatment with ABA (0.1‐1.0 m M ) under non‐inductive 8‐h short days (SD) resulted in cessation of leaf and tiller production and in the development of typical features of dormancy: bulbing at the base of the tillers and leaf senescence. Short‐term applications of ABA had similar effects but dormancy was transient, i.e. after a short while, leaf growth from the formed bulbs was resumed. ABA treatment of plants growing under an inductive 16‐h photoperiod (LD) enhanced the onset of dormancy. Endogenous levels of ABA in leaf blades and at the tiller base (where the bulb develops) increased markedly after the plants were transferred from SD to LD. This increase was greater in the tiller base, and concomitant with bulb maturation. High temperature (27/22 vs 22/17°C) accelerated both bulb development and ABA accumulation in leaf blades.
These results suggest that ABA plays a key role in the photoperiodic induction and development of summer‐dormancy in P. bulbosa .     

Inhibition of glucose transport by phenylglyoxal: Both dissipation of ΔpH and essential arginyl residues are involved

The effects of the arginine modifying reagent phenylglyoxal (PGO) on solute transport was studied in two cellular systems: protoplasts isolated from the mesophyll of Vicia faba L. and XD cell suspension culture of Nicotiana tabacum L. cv. Xanthi. The solutes in the case of the protoplasts were the non‐metabolizable glucose analog 3‐O‐methyl‐D‐glucose (MeG), and a non‐metabolizable amino acid analog α‐aminoisobutyric acid (AIB), whereas the solutes for the cell suspension were AIB and nitrate. Solute transport in both systems was rapidly inhibited by PGO. Exposure of the protoplasts to light enhanced the initial rate of MeG uptake. PGO rapidly inhibited MeG uptake in both the light and the dark, the half‐time for inactivation being less than 3 min. Flux analysis of double‐labeled MeG showed that initial MeG uptake was mediated mainly by the plasma membrane transport system and that it was inhibited by PGO. Maximal inhibition of initial MeG uptake rate was observed at PGO concentrations of 1 m M and above. PGO treatment altered rapidly the equilibrium distribution of the ΔpH probe dimethyloxazolidine (DMO) in both cellular systems, indicating dissipation of ΔpH between cell and medium. In the protoplasts, PGO inhibited both DMO and MeG uptake at pH 5.5; however, at pH 7.0, where ΔpH is minimal, only MeG uptake was inhibited. Our results suggest that PGO has two effects on glucose uptake: an indirect effect through ΔpH dissipation and a direct effect through interaction with essential arginyl residues in the glucose transporter.     

Gustatory responses to feeding‐ and non‐feeding‐stimulant chemicals, with an emphasis on amino acids, in rainbow trout

Specific receptor and fibre types of rainbow trout Oncorhynchus mykiss involved in the detection and discrimination of amino acids and a heterogeneous collection of compounds were investigated by recording the electrical activity of the maxillary branch of the facial nerve innervating taste buds inside the upper jaw. Proline (Pro), alanine (Ala), leucine (Leu), betaine (Bet) and 2‐amino‐3‐guanidinopropionic acid (Agp) were the major amino acids detected by the gustatory system. The two experimental approaches, concentration‐response curves and cross‐adaptations, showed that all amino acids were detected by three independent receptor types: Pro ‐, Agp/Bet ‐ and Leu ‐receptors. Bile acids, the most potent stimulants recorded, were detected by a single receptor type independent of those for amino acids, with threshold concentrations of 10⁻¹² M. Strychnine, quinine and tetrodotoxin may have partially shared a single receptor mechanism. The gustatory sensibility narrowly tuned towards the amino acid spectrum compared to those for a diverse array of non‐feeding stimulant chemicals, combined with feeding behaviour triggered primarily by vision and olfaction, suggest that in rainbow trout, and possibly other salmonid species, gustatory chemical cues, in addition to food finding and intake, play an important role in detecting poisonous prey and substances.