Mucin-associated T antigens in benign and malignant epithelium of the gall bladder,extrahepatic bile ducts and ampulla of Vater

The mucin-associated antigens Tn, sialosyl-Tn (STn), T and sialosyl-T (STAg) antigens accumulate through aberrant and incomplete glycosylation in malignant epithelial cells. Their diagnostic and prognostic significance in tumours of the colon and cervix has been described, and a possible role for Tn antigen in cell-to-cell adhesion has been suggested. These antigens have been demonstrated through peanut agglutinin (PNA) lectin binding and more recently using specific monoclonal antisera. Differences between the two methods have been described, which may be due to fixation schedules and/or specificity. We have investigated the effect of fixation on the binding of biotinylated PNA lectin and compared its reactivity with the immunoreactivity of monoclonal antisera to Tn, STn, T and STAg antigens in benign and malignant epithelium of the gall bladder, extrahepatic bile ducts and ampulla of Vater. We found that short-term fixation in formol sublimate resulted in poor PNA binding. All other tested fixation schedules showed strong perinuclear binding, similar to that found on cryostat sections. When compared with monoclonal antisera, PNA binding demonstrated the lowest specificity in benign epithelium. In both benign and malignant epithelium, the two methods cannot substitute for each other. STn and STAg antigens were found to be oncodevelopmental throughout the extrahepatic biliary tract. When used in a panel, they are useful as diagnostic markers of malignancy in gall bladder epithelium.     

Scaling of fast-start performance and its thermal dependence in mummichog Fundulus heteroclitus

Fast-start predator-escape performance and its sensitivity to temperature (24, 30, and 36°C) were evaluated in mummichog Fundulus heteroclitus across a range of body sizes spanning YOY to adult (35–68 mm standard length). Mummichogs exhibit isometry of body dimensions and areas of the dorsal and anal fins but negative allometry of the caudal fin area. These scaling relationships are consistent with observed decreases in fast-start angular velocities with increasing body size. Linear velocity, on the contrary, does not vary with size, and both large and small mummichogs are capable of traversing similar distances in a given amount of time. In addition, temperature influences fast-start performance in similar ways over the size range, though the magnitude of the effect varies with size for some performance measures. In general, fast-start performance increases with test temperature, but mummichogs acclimated to warmer temperatures exhibit lower performance at each test temperature. Altogether, our results suggest that mummichogs across the adult size range may suffer decreases in their predator-escape performance as increasing sea temperatures combine with short-term temperature fluctuations in the estuaries these fish occupy.     

Fast-start escape performance across temperature and salinity gradients in mummichog Fundulus heteroclitus

Fast-start predator-escape performance of mummichogs Fundulus heteroclitus was tested across field-informed variation in temperature (24, 30 and 36°C) and salinity (2, 12 and 32 ppt). Performance was similar across temperatures and salinities when fish were allowed to acclimate to these conditions. However, when mummichogs experienced acute temperature changes, performance exhibited thermal dependence in two contrasting ways. Fast-start turning rates and linear speeds varied directly with the temperature at which the manoeuvre was executed, but these aspects of performance varied inversely with acclimation temperature, with cool-acclimated fish exhibiting faster starts across test temperatures. Temperature effects were consistent across salinities. These results suggest that while mummichogs increase performance with acute temperature increases, long-term rises in sea temperature may cause these fish to become more susceptible to predation during abrupt cooling events, such as when storm events flood shallow water estuaries with cool rainwater.     

Rodlet cells in the posterior intestine of embryos and neonates of two poeciliid species

Rodlet cells occurred in the posterior intestine of embryos and neonates of the swordtail Xiphophorus nigrensis , its hybrids with Xiphophorus pygmaeus and in the platyfish Xiphophorus maculatus . This is the first observation of these enigmatic cells in a viviparous teleost prior to birth. This finding lends support to the endogenous tenet regarding the origin of this cell.     

The effect of acute warming and thermal acclimation on maximum heart rate of the common killifish Fundulus heteroclitus

Common killifish Fundulus heteroclitus were acclimated to ecologically relevant temperatures (5, 15 and 33°C) and their maximum heart rate (f_Hmax) was measured at each acclimation temperature during an acute warming protocol. Acclimation to 33°C increased peak f_Hmax by up to 32% and allowed the heart to beat rhythmically at a temperature 10°C higher when compared with acclimation to 5°C. Independent of acclimation temperature, peak f_Hmax occurred about 3°C cooler than the temperature that first produced cardiac arrhythmias. Thus, when compared with previously published values for the critical thermal maximum of F. heteroclitus, the temperature for peak f_Hmax was cooler and the temperature that first produced cardiac arrhythmias was similar to these critical thermal maxima. The considerable thermal plasticity of f_Hmax demonstrated in the present study is entirely consistent with eurythermal ecology of killifish, as shown previously for another eurythermal fish Gillichthys mirabilis.     

Temporal dynamics in growth and white skeletal muscle composition of the mummichog Fundulus heteroclitus during chronic hypoxia and hyperoxia

Specific growth rate (G(S) ) and white skeletal muscle composition were measured in the mummichog Fundulus heteroclitus over a period of 28 days at four levels of dissolved oxygen (DO): severe hypoxia (c. 1.2 mg O(2) l(-1) ), moderate hypoxia (3.0 mg O(2) l(-1) ), normoxia (7.1 mg O(2) l(-1) ) and hyperoxia (10.6 mg O(2) l(-1) ). The G(S) was calculated over 0-8, 0-14, 0-28 and 14-28 days, and muscle protein, lactate dehydrogenase (LDH), DNA, RNA and water were measured at 0, 8, 14 and 28 days. Exposure of fish to severe hypoxia was associated with significantly reduced G(S) , lower muscle protein content and lower RNA:DNA compared with other DO treatments. When calculated over the first and second half of the 28 day exposure, however, G(S) of fish in severe hypoxia increased significantly during the second two-week interval, to the same rate as that of normoxic fish. Muscle LDH activity and water content were not significantly affected by DO level. Neither moderate hypoxia nor hyperoxia significantly affected G(S) or any biochemical variable. The results demonstrate that F. heteroclitus can tolerate wide variation in ambient oxygen concentration and, during prolonged exposure to severe hypoxia, shows significant compensation for the initial negative effects on growth. The capacity of F. heteroclitus to grow over a wide range of DO probably contributes to its ability to exploit habitats characterized by marked variation in oxygen availability.     

Asian chestnut gall wasp in Tuscany: gall characteristics,egg distribution and chestnut cultivar susceptibility

• 1 Preliminary investigations were carried out on Dryocosmus kuriphilus Yasumatsu on Castanea sativa Miller in Tuscany to assess variations in gall characteristics in coppice and high forest at two crown heights (height < 2 or 2–6 m), influence of bud size and bud position on oviposition rates and susceptibility of three cultivars.
• 2 Gall size may depend on various factors, including wasp population density. In the present study area, small galls (with one or two cells) were the most numerous in 2008, whereas larger galls (with more than three cells) prevailed in 2009.
• 3 Dryocosmus kuriphilus oviposition occurrence was influenced by both bud size and bud position. Buds with eggs tended to be larger in size compared with bud without eggs, suggesting that D. kuriphilus females prefer to lay eggs in larger buds (approximately 6 mm³) compared with smaller buds (approximately 3 mm³). The mean number of eggs per bud tended to decrease from the apical bud toward the basal bud.
• 4 Three C. sativa cultivars, Carpinese, Fusca and Cesurone, were examined. Fusca grafts had significantly more galls compared with Carpinese and Cesurone, whereas Cesurone grafts had more larvae per bud compared with Carpinese and Fusca. Overall, the Carpinese cultivar may be less susceptible to D. kuriphilus galling compared with the Fusca and Cesurone cultivars.

     

Ca2+ transport by opercular epithelium of the fresh water adapted euryhaline teleost,Fundulus heteroclitus

We examined transepithelial transport of Ca²⁺ across the isolated opercular epithelium of the euryhaline killifish adapted to fresh water. The opercular epithelium, mounted in vitro with saline on the serosal side and fresh water (0.1 mmol·l^–1 Ca²⁺) bathing the mucosal side, actively transported Ca²⁺ in the uptake direction; net flux averaged 20–30 nmol·cm^–2·h^–1. The rate of Ca²⁺ uptake varied linearly with the density of mitochondria-rich cells in the preparations. Ca²⁺ uptake was saturable, apparent K _1/2 of 0.348 mmol·l^–1, indicative of a multistep transcellular pathway. Ca²⁺ uptake was inhibited partially by apically added 0.1 mmol·l^–1 La³⁺ and 1.0 mmol·l^–1 Mg²⁺. Addition of dibutyryl-cyclic adenosine monophosphate (0.5 mmol·l^–1)+0.1 mmol·l^–1 3-isobutyl-l-methylxanthine inhibited Ca²⁺ uptake by 54%, but epinephrine, clonidine and isoproterenol were without effect. Agents that increase intracellular Ca²⁺, thapsigargin (1.0 mol·l^–1, serosal side), ionomycin (1.0 mol·l^–1, serosal side) and the calmodulin blocker trifluoperazine (50 mol·l^–1, mucosal side) all partially inhibited Ca²⁺ uptake. In contrast, apically added ionomycin increased mucosal to serosal unidirectional Ca²⁺ flux, indicating Ca²⁺ entry across the apical membrane is rate limiting in the transport. Verapamil (10–100 mol·l^–1, mucosal side), a Ca²⁺ channel blocker, had no effect. Results are consistent with a model of Ca²⁺ uptake by mitochondria rich cells that involves passive Ca²⁺ entry across the apical membrane via verapamil-insensitive Ca²⁺ channels, intracellular complexing of Ca²⁺ by calmodulin and basolateral exit via an active transport process. Increases in intracellular Ca²⁺ invoke a downregulation of transcellular Ca²⁺ transport, implicating Ca²⁺ as a homeostatic mediator of its own transport.Abbreviations DASPEI 2-(4-dimethylaminostyryl)-N-ethylpyridinium iodide - db-cAMP dibutyryl-cyclic adenosine monophosphate - FW fresh water - G _t transepithelial conductance - I _sc short-circuit current - IBMX 3-isobutyl-1-methylxanthine - SW sea water - TFP trifluoperazine - V _t transepithelial potential     

Characterization of two Ca-ATPases in gill epithelium from the killifish (Fundulus heteroclitus)

1. Two Ca-ATPases in the gill microsomal fraction from the killifish (Fundulus heteroclitus) have been characterized. 2. A (Ca2+ + Mg2+)-ATPase which has a high affinity for Ca2+, requires Mg2+ for activity and may be stimulated by calmodulin. 3. A (Ca2+ + Na+)-ATPase which has a low affinity for Ca2+ requires Na+ for activity, does not require Mg2+ and is probably not stimulated by calmodulin. 4. These enzymes may play a physiological role in killifish calcium regulation.     

Na+-dependent Ca2+ uptake in isolated opercular epithelium of Fundulus heteroclitus

It is concluded that Ca²⁺ transport across the basolateral membranes of the ionocytes in killifish skin is mediated for the major part by a Na⁺/Ca²⁺-exchange mechanism that is driven by the (transmembrane) Na⁺ gradient established by Na⁺/K⁺-ATPase. The conclusion is based, firstly, on the biochemical evidence for the presence of a Na⁺/Ca²⁺-exchanger next to the Ca²⁺-ATPase in the basolateral membranes of killifish gill cells. Secondly, the transcellular Ca²⁺ uptake measured in an Ussing chamber setup was 85% and 80% reduced in freshwater (FW) and SW (SW) opercular membranes, respectively, as the Na⁺ gradient across the basolateral membrane was directly or indirectly (by ouabain) reduced. Thapsigargin or dibutyryl-cAMP/IBMX in SW opercular membranes reduced Ca²⁺ influx to 46%, comparable to the effects seen in FW membranes [reduction to 56%; Marshall et al. 1995a]. Basal Ca²⁺ influx across the opercular membrane was 48% lower in membranes from fish adapted to SW than in membranes from fish adaptated to FW. Branchial Na⁺/K⁺-ATPase activity was two times higher in SW adapted fish. Accepted: 29 October 1996     

Actin microfilaments in melanophores of Fundulus heteroclitus

Summary In melanophores of Fundulus heteroclitus, hormone-stimulated melanosome aggregation is accompanied by cytoplasmic flow from the cellular processes to the perikaryon, and reversal of these events takes place upon hormone-induced melanosome dispersion. These cells contain parallel arrays of microtubules, the majority of which are located in the perikaryon and in cortical regions of the processes. Studies with heavy meromyosin binding demonstrated two types of actin filaments: 1) a decorated meshwork of filaments similar to those usually found in close association with plasma membranes, and 2) filaments decorated in a manner similar to that of stress fibers. There is an apparent increase in the amount of filaments during melanosome aggregation. These results are discussed in relation to intracellular movement.Supported, in part, by grants AM-5384 and AM-13724 from U.S.P.H.S., and grant 234046 from the Japanese Ministry of Education     

Control of Cl- transport in the operculum epithelium of Fundulus heteroclitus: long- and short-term salinity adaptation

The eurohaline fish, Fundulus heteroclitus, adapts rapidly to enhanced salinity by increasing the ion secretion by gill chloride cells. An increase of approximately 70 mOsm in plasma osmolarity was previously found during the transition. To mimic this in vitro, isolated opercular epithelia of seawater-adapted Fundulus mounted in a modified Ussing chamber were exposed to an increase in NaCl and/or osmolarity on the basolateral side, which immediately increased I(SC). Various Cl(-) channel blockers as well as the K(+) channel blocker Ba(2+) added to the basolateral side all inhibited the steady-state as well as the hypertonic stimulation of I(SC). The exists -agonist isoproterenol stimulates I(SC) in standard Ringer solutions. In contrast, when cell volume was kept at the larger value by simultaneous addition of water, the stimulation with isoproterenol was abolished, suggesting that the key process for activation of the Na(+), K(+), 2Cl(-) cotransporter is cell shrinkage. The protein kinase C (PKC) inhibitor chelerythrine and the myosin light chain kinase (MLCK) inhibitor ML-7 had strong inhibitory effects on the mannitol activation of I(SC), thus both MLCK and PKC are involved. The two specific protein kinase A (PKA) inhibitors H-89 and KT 5720 had no effect after mannitol addition whereas isoproterenol stimulation was completely blocked by H-89. This indicates that PKA is involved in the activation of the apical Cl(-) channel via c-AMP whereas the shrinkage activation of the Na(+), K(+), 2Cl(-) cotransporter is independent of PKA activation. The steady-state Cl(-) secretion was stimulated by an inhibitor of serine/threonine phosphatases of the PP-1 and PP-2A type and inhibited by a PKC inhibitor but not by a PKA inhibitor. Thus, it seems to be determined by continuous phosphorylation and dephosphorylation involving PKC but not PKA. The steady-state Cl(-) secretion and the maximal obtainable Cl(-) secretion were measured in freshwater-adapted fish and in fish retransferred to saltwater. No I(SC) could be measured in freshwater-adapted fish or in the fish within the first 18 h after transfer to saltwater. As evidenced from Western blot analysis using antiserine-antibodies, a heavily serine phosphorylated protein of about 190 kDa was consistently observed in the saltwater-acclimated fish, but was only weakly present in freshwater-acclimated fish. This observation indicates that acclimatization to saltwater stimulates the expression of this 190-kDa protein and/or a serine/threonine kinase, which subsequently phosphorylates the protein.     

Structural diversity of occluding junctions in the low-resistance chloride-secreting opercular epithelium of seawater-adapted killifish (Fundulus heteroclitus)

The structural features of the chloride-secreting opercular epithelium of seawater-adapted killifish (Fundulus heteroclitus) were examined by thin-section and freeze-fracture electron microscopy, with particular emphasis on the morphological appearance of occluding junctions. This epithelium is a flat sheet consisting predominantly of groups of mitochondriarich chloride cells with their apices associated to form apical crypts. These multicellular groups are interspersed in an otherwise continuous pavement cell epithelial lining. The epithelium may be mounted in Ussing-type chambers, which allow ready access to mucosal and serosal solutions and measurement of electrocal properties. The mean short-circuit current, potential difference (mucosal-side negative), and DC resistance for 19 opercular epithelia were, respectively, 120.0 +/- 18.2 microA/cm2, 12.3 +/- 1.7 mV, and 132.5 +/- 26.4 omega cm2. Short-circuit current, a direct measure of Cl- transport, was inhibited by ouabain (5 micron) when introduced on the serosal side, but not when applied to the mucosal side alone. Autoradiographic analysis of [3H]-ouabain-binding sites demonstrated that Na+,K+-ATPase was localized exclusively to basolateral membranes of chloride cells; pavement cells were unlabeled. Occluding junctions between adjacent chloride cells were remarkably shallow (20-25 nm), consisting of two parallel and juxtaposed junctional strands. Junctional interactions between pavement cells or between pavement cells and chloride cells were considerably more elaborate, extending 0.3-0.5 micron in depth and consisting of five or more interlocking junctional strands. Chloride cells at the lateral margins of crypts make simple junctional contacts with neighboring chloride cells and extensive junctions with contiguous pavement cells. Accordingly, in this heterogeneous epithelium, only junctions between Na+,K+-ATPase- rich chloride cells are shallow. Apical crypts may serve, therefore, as focal areas of high cation conductivity across the junctional route. This view is consistent with the electrical data showing that transmural resistance across the opercular eptihelium is low, and with recent studies demonstrating that transepithelial Na+ fluxes are passive. The simplicity of these junctions parallels that described recently for secretory cells of avian salt gland (Riddle and Ernst, 1979, J. Membr. Biol., 45:21-35) and elasmobranch rectal gland (Ernst et al., 1979, J. Cell Biol., 83:(2, Pt. 2):83 a[Abstr.]) and lends morphological support to the concept that paracellular ion permeation plays a central role in ouabain-sensitive transepithelial NaCl secretion.     

The fine structure of the gall bladder epithelium of the sheep

Summary The electron microscopy of the gall bladder epithelium in the sheep shows that the cells are secretory. They have an extensive Golgi apparatus and sparse endoplasmic reticulum with secretory droplets localised in the apical region and have been referred to the group known as mucoid cells. The intercellular spaces which are elaborately developed in those species whose gall bladder is primarily absorptive are poorly developed. Pinocytosis is not a prominent feature. It is believed that the features noted are correlated with the reported absence of absorption in the ungulate gall bladder.     

Histochemical and biochemical studies of carbonic anhydrase activity in the opercular epithelium of the euryhaline teleost, Fundulus heteroclitus

Carbonic anhydrase (CAH) activity was biochemically measured and histochemically localized (at both the light and electron microscope levels) in isolated opercular membranes from teleost fish, Fundulus heteroclitus, adapted to freshwater (FW), seawater (SW), and double-strength seawater (2 x SW). The normal morphology of this membrane showed that its epithelial portion consisted of five cell types: (1) chloride cells, which have been previously implicated as responsible for the active chloride transport across the epithelium; (2) mucous cells; (3) pavement cells, which formed the major portion of the free epithelial surface; (4) supportive cells, which had an abundance of intermediate (10 nm)-type filaments suggesting a structural role for these cells; and (5) vesicular cells, which were characterized by various types of membrane-bound vesicles, including lysosomes, and numerous free ribosomes. Vesicular cells may be stem cells and/or endocrine cells. Hansson's histochemical method for CAH revealed cobalt sulfide reaction product confined to the following structures in fish from each environment: (1) chloride cells: throughout the cytoplasm and some nuclear staining; (2) mucous cells: throughout the cytoplasm, some nuclear staining, and some in mucous granules; (3) vesicular cells: confined to lysosomes, some of the vesicles, and nucleoli; (4) a small portion of the intracellular space between adjacent vesicular cells and supportive cells; and (5) supportive cells: in nucleoli and occasionally in larger membrane-bound lysosomelike structures. Acetazolamide (10(-5) M) and potassium cyanate (KCNO) (10(-1) M) in Hansson's incubation medium completely inhibited the formation of reaction product. Biochemical determination of CAH activity on vascularly perfused, isolated opercular membranes showed no statistically significant difference in enzyme activity between environmental groups. The following units of activity/mg opercular membrane protein were measured: FW: 0.63 +/- 0.02; SW: 0.43 +/- 0.08; 2 x SW: 0.64 +/- 0.09.     

Sulfide-hemoglobin interactions in the sulfide-tolerant salt marsh resident,the California killifish Fundulus parvipinnis

Summary Sulfide can potentially damage hemoglobin or be detoxified by hemoglobin. In the sulfide-tolerant California killifish neither seems to be the case at environmentally realistic (micromolar) and physiologically relevant (millimolar) sulfide concentrations. An 8-h exposure of killifish to 5 and 8 mmol sulfide · 1^-1 results in 50–100% mortality, but not due to sulfhemoglobin (where sulfide covalently binds to the porphyrin) nor ferric hemoglobin (Hb⁺), both dysfunctional hemoglobin derivatives. Killifish hemoglobin converts to sulfhemoglobin in vitro only in the presence of 1–5 mmol sulfide · 1^-1. The amount of sulfhemoglobin formed increases with time and heme concentration but decreases with pH. Hb⁺ binds sulfide as ferric hemoglobin sulfide (Hb⁺S, an unstable complex where sulfide ligates to the iron), and also as sulfhemoglobin. Killifish blood does not catalyze the oxidation of 10–500 mol sulfide · 1^-1 to any appreciable extent. Radiolabeled sulfide incubated with oxyhemoglobin or whole blood disappears at rates greater than in buffers, but only minimal amounts of thiosulfate and no sulfate nor sulfite are formed (elemental sulfur and bound sulfide not quantified). Sulfide disappearance rates increase linearly with initial sulfide concentration. Hb⁺ does catalyze the oxidation of sulfide to thiosulfate in vitro. Similar experiments on another sulfide-tolerant species, the long-jawed mudsucker Gillichthys mirabilis, produced similar results.Abbreviations ANOVA analysis of variance - BV benzyl viologen - HEPES N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid - HPLC high-pressure liquid chromatography - RBC red blood cells - SHb sulfhemoglobin     

Tolerance to Environmental Contaminants in the Mummichog,Fundulus heteroclitus

Mummichogs or killifish (Fundulus heteroclitus) are abundant estuarine fish that can tolerate widely varying environmental conditions. They are found in some highly contaminated sites, and their development of tolerance to toxicants has been studied. Populations have developed resistance to methylmercury, kepone, dioxins, polychlorinated biphenyls, and polyaromatic hydrocarbons. This article describes what is known about their tolerance, and discusses similarities and differences among these resistant populations. In some cases tolerance is seen only in early life stages, while in other cases tolerance is seen in adults also. In many of the populations, adults show signs of stress. The mechanism of embryo tolerance to meHg appears to be reduced chorionic permeability, and more rapid development through sensitive stages. One mechanism of resistance to dioxins, PCBs and PAHs is non-responsiveness (lack of inducibility) of CYP1A. In some populations it is already elevated, while in others it is not. Another mechanism in the PAH-resistant fish is elevation of the phase II metabolizing enzyme, GST. The tolerance appears to be genetic. In populations in which it has been studied, the age structure is skewed towards younger fish, and adults appear to put more energy into reproduction as a way of maintaining the population in the stressful environment.