Application of the general fluid circuit theory to the chloride-bromide-deuterium oxide experiment

The general fluid circuit theory of active chloride absorption is applied to an experiment in which chloride, bromide, and deuterium oxide were simultaneously absorbed from the lower ileum, chloride only moving against a concentration gradient. The conclusion is reached that the constant for active absorption of deuterium oxide is equal, within reasonable limits of error, to the constant for active chloride absorption in accordance with the assumption that the absorption of water carries chloride out of the intestinal lumen without changing its concentration.     

Aerobic mineralization of 4,4′-dichlorobiphenyl and 4-chlorobenzoic acid by a novel natural bacterial strain that grows poorly on benzoate and biphenyl

Achromobacter xylosoxidans strain IR08 was isolated from soil contaminated with electrical transformer fluid by enrichment culture containing Aroclor 1221 as the sole carbon source. This strain was found to grow on all monochlorobiphenyls, 4,4′-dichlorobiphenyl (4,4′-diCB) and a wide range of other xenobiotic compounds. During growth on 4,4′-diCB, a near-stoichiometric amount of chloride was excreted into the culture fluid in less than 5 days and growth yield was more than twice that achieved on biphenyl. The production of 4-CBA or chlorocatechol as a metabolite was not observed. Quite unusually, coincubation of strain IR08 with 4,4′-diCB and biphenyl at relatively equal concentrations showed preferential utilization of the chlorobiphenyl: 4,4′-diCB was mineralized in less than 5 days concomitant with stoichiometric release of chloride, while biphenyl was poorly degraded. Growth on 2.5 mM CBA also resulted in complete disappearance of the substrate, however, inorganic chloride recovered from the culture broth was less than 65%. The isolation of a dichlorobiphenyl-mineralizing rather than transformation strain such as IR08 is an important advance in an effort to develop effective bioremediation strategy for polychlorinated biphenyl-contaminated soil.     

Identification,release and olfactory detection of bile salts in the intestinal fluid of the Senegalese sole (<Emphasis Type="Italic">Solea senegalensis</Emphasis>)

Olfactory sensitivity to bile salts is wide-spread in teleosts; however, which bile salts are released in sufficient quantities to be detected is unclear. The current study identified bile salts in the intestinal and bile fluids of Solea senegalensis by mass spectrometry–liquid chromatography and assessed their olfactory potency by the electro-olfactogram. The main bile salts identified in the bile were taurocholic acid (342 mM) and taurolithocholic acid (271 mM) plus a third, unidentified, bile salt of 532.3 Da. These three were also present in the intestinal fluid (taurocholic acid, 4.13 mM; taurolithocholic acid, 0.4 mM). In sole-conditioned water, only taurocholic acid (0.31 μM) was released in sufficient quantities to be measured (release rate: 24 nmol kg⁻¹ min⁻¹). Sole had high olfactory sensitivity to taurocholic acid but not to taurolithocholic acid. Furthermore, olfactory sensitivity was higher in the upper (right) olfactory epithelium than the lower (left). These two bile acids contribute about 40% of the olfactory potency of intestinal fluid and account for the difference in potency at the two epithelia. Taurocholic acid (but not taurolithocholic acid), and possibly other types of bile acid not tested, could be used as chemical signals and the upper olfactory epithelium is specialised for their detection.     

Enterocyte chloride and water secretion into the small intestine after enterotoxin challenge: Unifying hypothesis or intellectual dead end?

Many forms of diarrhoeal disease, particularly so called “secretory” diarrhoeal disease are thought to arise by the active secretion of chloride ion from the enterocytes, creating an osmotic gradient for fluid movement into the small intestinal lumen. This model implies that normally occurring intestinal secretion is catastrophically enhanced by bacterial enterotoxins. This review advocates that neither normal nor abnormal intestinal secretion from the enterocytes occurs and that no competent proof for chloride secretion exists. Prior to 1970, the physiological evidence failed to support the concept of the formation of intestinal juice as a normal intestinal event. support the concept of the formation of intestinal juice as a normal intestinal event. The concept was later revived to explain the high rate of fluid entry into the lumen after exposure to cholera toxin. Much evidence has been advanced for the chloride secretion hypothesis, the dominant secretory paradigm after 1974, but is the evidence sufficiently compelling for it to be regarded as proving the chloride secretory model? The evidence falls into four categories and a fifth conjectural argument that proposes that an abnormal chloride ion channel in cystic fibrotic sufferers confers a natural selective advantage by preventing diarrhoeal disease. Secretion is putatively demonstrated by 1) showing that mass transfer of fluid is into the lumen (secretion) and not merely a failure to transport out of the lumen (failed absorption). Support is offered by 2) chloride ion flux measurementsin vitro in Ussing chambers and by 3) shortcircuit current measurements that are consistent with and purport to show chloride ion movement into the lumen. In addition, 4) pharmacological agents are identified that affect short-circuit current and these are assumed to be anti-secretory, consistent with the biochemical mechanism for secretion, confirmed wherever possible by mouse knock-out models. Finally, the proxy methods used to study water movement such as elevated short-circuit current measurements show these to be absent in cystic fibrotic patients. The enterocyte secretion hypothesis is challenged here on the basis of an examination of the methods used to show secretion, particularly after exposing the small intestine to heat stable enterotoxin (STa) fromE. coli. STa is thought to be secretory because fluid entry into the lumen is claimed, enhanced isotopic flux of chloride ion towards the lumen occurs, an increase in short-circuit current is found, preventable by various drugs that are deemed likely to be anti-secretory and also because the short-circuit current changes after STa are not seen in cystic fibrotic patients. Using volume recoveryin vivo, STa is found not to be secretory but only anti-absorptive. Hence, other techniques used to show secretion are not fit for that purpose. If STa is identified as secretory and yet no secretion occurs, how reliable is the evidence for other toxins being secretory when these methods are used? This review concludes that chloride ion secretion is unproven. A review of the literature indicates that secretion occurs not because epithelial cells actively pump water but by interdiction of fluid absorption, increased conductivity through tight junctions and an increased hydrostatic driving force through elevated capillary pressure. The exclusive focus on chloride secretion may explain the failure to develop antisecretory drugs over the last three decades.     

Hyperacidity of secreted fluid from submucosal glands in early cystic fibrosis

Prior studies have shown that fluid secretions from airway submucosal glands in cystic fibrosis (CF) are reduced and hyperviscous, possibly contributing to the pathogenesis of CF airway disease. Because the CF transmembrane conductance regulator (CFTR) protein can transport both chloride and bicarbonate, we investigated whether gland fluid pH is abnormal in early CF, using nasal biopsies from pediatric subjects having minimal CF lung disease. Gland fluid pH, measured in freshly secreted droplets under oil stained with BCECF-dextran, was 6.57 ± 0.09 (mean ± SE) in biopsies from six CF subjects, significantly lower than 7.18 ± 0.06 in eight non-CF biopsies (P < 0.01). To rule out the possibility that the apparent gland fluid hyperacidity in CF results from modification of fluid pH by the airway surface, a microcannulation method was used to measure pH in fluid exiting gland orifices. In pig trachea and human bronchi, gland fluid pH was reduced by up to 0.45 units by CFTR inhibitors, but was not affected by amiloride. Acid base transport in the surface epithelium of pig trachea was studied from pH changes in 300-nl fluid droplets deposited onto the oil-covered airway surface. The droplets had specified ionic composition/pH and/or contained transporter activators/inhibitors. We found evidence for CFTR-dependent bicarbonate transport by the tracheal surface epithelium as well as ATP/histamine-stimulated proton secretion, but not for sodium/proton or chloride/bicarbonate exchange. These results provide evidence for intrinsic hyperacidity in CF gland fluid secretions, which may contribute to CF airway pathology. cystic fibrosis transmembrane conductance regulator; airway; fluorescence microscopy; pH regulation     

Intercalation compounds of hydrotalcite-like anionic clays with anti-inflammatory agents,II: Uptake of diclofenac for a controlled release formulation

The purpose of this study was to investigate whether hydrotalcite is able to intercalate diclofenac, a nonsteroidal anti-inflammatory drug, and release it in a controlled manner. Layered Mg−Al hydrotalcite in the chloride form was used as a host, and the intercalation compound was prepared by Cl⁻/diclofenac anionic exchange. Drug release from the intercalation compound was performed in vitro in simulated intestinal fluid at pH 7.5 according to USP 24 and in a pH 7.0 solution designed to mimic the ionic conditions of the small intestine. Results from the intercalation process show that hydrotalcite is able to intercalate diclofenac with a simple procedure and with a good drug loading (55% wt/wt). The in vitro drug release was remarkably lower than that from the corresponding physical mixture at both pH 7.5 and pH 7.0. In the latter case, the release was not complete at 24 hours. The kinetic analysis shows the importance of the diffusion through the particle in controlling the drug release rate. The obtained results show that hydrotalcite may be used to prepare modified release formulations.     

Bioavailability Study of Berberine and the Enhancing Effects of TPGS on Intestinal Absorption in Rats

Berberine chloride (BBR) is a natural isoquinoline alkaloid extracted from medicinal herbs. It has been reported that the intestinal absorption of BBR is very low. In this study, the absolute bioavailability of BBR was studied, and the enhancing effects of d-α-tocopheryl polyethylene glycol 1000 succinate (TPGS) on intestinal absorption were investigated in rats. BBR injection was administrated via the femoral vein at a dose of 1.0 mg kg⁻¹ in intravenous group, and BBR oral formulations were administrated by oral gavage at a dose of 100 mg kg⁻¹ in BBR control (control) group and BBR-TPGS (test) group, respectively. The result showed that BBR had a very low absolute bioavailability of 0.68%, and TPGS could enhance intestinal absorption of BBR significantly. TPGS at a concentration of 2.5% could improve peak concentration (C _max) and area under the curve (AUC_0–36) of BBR by 2.9 and 1.9 times, respectively. The absorption enhancing ability of TPGS may be due to its ability to affect the biological activity of P-glycoprotein and thereby reduce the excretion of absorbed BBR into the intestinal lumen. This study indicated that absolute bioavailability of BBR was 0.68% in rats, and TPGS was a good absorption enhancer capable of enhancing intestinal absorption of BBR significantly.     

Lipopolysaccharide of Escherichia coli, polyamines, and acetic acid stimulate cell proliferation in intestinal epithelial cells

Summary Our aim was to examine whether lipopolysaccharide of Escherichia coli, polyamines of dietetic and/or bacterial origin, and products of the bacterial metabolism influence cell proliferation in epithelial cells from the colon and small intestine. Lipopolysaccharide of Escherichia coli 0111:B4 was incubated with cultures from human colonic mucosa. The mitoses were arrested with Vincristine and the total number of metaphases per crypt was counted. In addition, lipopolysaccharide was incubated with a human colonic epithelial cell line from adenocarcinoma (LS-123 cells) and with a nontransformed small intestinal cell line from germ-free rats (IEC-6 cells) for 24 h. In the last 4 h, the cells were labeled with tritiated thymidine. The cells were incubated with putrescine, cadaverine, and spermidine at 10⁻¹¹–10⁻³ M and with acetic acid (10⁻⁵–10⁻¹ M), acetaldehyde (10⁻¹⁰–10⁻⁴ M) and ammonium chloride (1–20 mM). Lipopolysaccharide of Escherichia coli increased the number of arrested metaphases in human colonic crypts and DNA synthesis in L-123 and IEC-6 cells (P<0.001). All polyamines increased DNA synthesis in the colonic and small intestinal cell lines, the effects being more marked for putrescine (P<0.001). The higher concentrations of acetic acid increased DNA synthesis in both epithelial cell lines (P<0.001). Acetaldehyde slightly decreased DNA synthesis in LS-123 cells at cytotoxic concentrations. Ammonium chloride did not significantly affect DNA synthesis. The final concentration of nonionized ammonia was less than 3%. It is concluded that lipopolysaccharides of Escherichia coli and intraluminal factors derived from microorganisms increase cell proliferation in human colonic crypts and intestinal epithelial cell lines.     

cAMP-dependent exocytosis and vesicle traffic regulate CFTR and fluid transport in rat jejunum in vivo

The cystic fibrosis transmembraneconductance regulator (CFTR) channel is regulated by cAMP-dependentvesicle traffic and exocytosis to the apical membrane in some celltypes, but this has not been demonstrated in the intestinal crypt. Thedistribution of CFTR, lactase (control), and fluid secretion weredetermined in rat jejunum after cAMP activation in the presence ofnocodazole and primaquine to disrupt vesicle traffic. CFTR and lactasewere localized by immunofluorescence, and surface proteins weredetected by biotinylation of enterocytes. Immunoprecipitates frombiotinylated and nonbiotinylated cells were analyzed by streptavidindetection and immunoblots. Immunolocalization confirmed acAMP-dependent shift of CFTR but not lactase from a subapicalcompartment to the apical surface associated with fluid secretion thatwas reduced in the presence of primaquine and nocodazole. Analysis ofimmunoblots from immunoprecipitates after biotinylation revealed a3.8 ± 1.7-fold (P < 0.005) increase ofsurface-exposed CFTR after vasoactive intestinal peptide (VIP). Thesemeasurements provide independent corroboration supporting a role forvesicle traffic in regulating CFTR and cAMP-induced fluid transport inthe intestine.

     

Ecto-5′-nucleotidase and intestinal ion secretion by enteropathogenic Escherichia coli

Enteropathogenic Escherichia coli (EPEC) triggers a large release of adenosine triphosphate (ATP) from host intestinal cells and the extracellular ATP is broken down to adenosine diphosphate (ADP), AMP, and adenosine. Adenosine is a potent secretagogue in the small and large intestine. We suspected that ecto-5′-nucleotidase (CD73, an intestinal enzyme) was a critical enzyme involved in the conversion of AMP to adenosine and in the pathogenesis of EPEC diarrhea. We developed a nonradioactive method for measuring ecto-5′-nucleotidase in cultured T84 cell monolayers based on the detection of phosphate release from 5′-AMP. EPEC infection triggered a release of ecto-5′-nucleotidase from the cell surface into the supernatant medium. EPEC-induced 5′-nucleotidase release was not correlated with host cell death but instead with activation of phosphatidylinositol-specific phospholipase C (PI-PLC). Ecto-5′-nucleotidase was susceptible to inhibition by zinc acetate and by α,β-methylene-adenosine diphosphate (α,β-methylene-ADP). In the Ussing chamber, these inhibitors could reverse the chloride secretory responses triggered by 5′-AMP. In addition, α,β-methylene-ADP and zinc blocked the ability of 5′-AMP to stimulate EPEC growth under nutrient-limited conditions in vitro. Ecto-5′-nucleotidase appears to be the major enzyme responsible for generation of adenosine from adenine nucleotides in the T84 cell line, and inhibitors of ecto-5′-nucleotidase, such as α,β-methylene-ADP and zinc, might be useful for treatment of the watery diarrhea produced by EPEC infection.     

Activation of AMPK Inhibits Cholera Toxin Stimulated Chloride Secretion in Human and Murine Intestine

Increased intestinal chloride secretion through chloride channels, such as the cystic fibrosis transmembrane conductance regulator (CFTR), is one of the major molecular mechanisms underlying enterotoxigenic diarrhea. It has been demonstrated in the past that the intracellular energy sensing kinase, the AMP-activated protein kinase (AMPK), can inhibit CFTR opening. We hypothesized that pharmacological activation of AMPK can abrogate the increased chloride flux through CFTR occurring during cholera toxin (CTX) mediated diarrhea.Chloride efflux was measured in isolated rat colonic crypts using real-time fluorescence imaging. AICAR and metformin were used to activate AMPK in the presence of the secretagogues CTX or forskolin (FSK). In order to substantiate our findings on the whole tissue level, short-circuit current (SCC) was monitored in human and murine colonic mucosa using Ussing chambers. Furthermore, fluid accumulation was measured in excised intestinal loops.CTX and forskolin (FSK) significantly increased chloride efflux in isolated colonic crypts. The increase in chloride efflux could be offset by using the AMPK activators AICAR and metformin. In human and mouse mucosal sheets, CTX and FSK increased SCC. AICAR and metformin inhibited the secretagogue induced rise in SCC, thereby confirming the findings made in isolated crypts. Moreover, AICAR decreased CTX stimulated fluid accumulation in excised intestinal segments.The present study suggests that pharmacological activation of AMPK effectively reduces CTX mediated increases in intestinal chloride secretion, which is a key factor for intestinal water accumulation. AMPK activators may therefore represent a supplemental treatment strategy for acute diarrheal illness.     

Subcellular distribution of CFTR in rat intestine supports a physiologic role for CFTR regulation by vesicle traffic

The cystic fibrosis transmembrane conductance regulator (CFTR) is a cAMP-activated chloride channel critical to intestinal anion secretion. In addition to phosphorylation, vesicle traffic regulates CFTR in some epithelial cells. Studies of cultured intestinal cells are conflicting regarding the role of cAMP-dependent vesicle traffic in regulating chloride transport. Whether CFTR is present in vesicular compartments within chloride secretory cells in the intestine is unknown and the role of cAMP-dependent vesicle insertion in regulating CFTR and intestinal fluid secretion remains unclear. The purpose of this study was to: (1) examine and quantify the subcellular distribution for CFTR in rat intestine, (2) further define the ultrastructure of the previously identified CFTR High Expresser (CHE) cell, and (3) examine the cellular distribution of CFTR following cAMP stimulation in vivo. Using the sensitive techniques of cryoimmunogold electron microscopy we identified CFTR in subapical vesicles and on the apical plasma membrane in crypt, Brunner glands, and CHE cells. cAMP stimulation in rat proximal small intestine produced a fluid secretory response and was associated with an apical redistribution of CFTR, supporting a physiologic role for cAMP-dependent CFTR vesicle insertion in regulating CFTR in the intestine.     

Relative amounts of sialic acid and fucose of amniotic fluid glycoconjugates in relation to pregnancy age

The present knowledge concerning the glycan structures and role of glycoconjugates derived from amniotic fluid is fragmentary and mainly focuses on the individual glycoproteins. The question has arisen as whether the general glycosylation pattern of amniotic fluid glycoconjugates can change with the progression of a normal pregnancy. In the present work we have described the dynamic, quantitative alterations in relative amounts of sialic acid and fucose linked by a variety of anomeric linkages to subterminal oligosaccharide structures of amniotic fluid glycoconjugates in relation to pregnancy age. The analysis was performed in the following groups of amniotic fluids derived from normal pregnancy by lectin dotting method: “2nd trimester” (14–19 weeks), “3rd trimester” (29–37 weeks), “perinatal period” (38–40 weeks) , “delivery at term” (39–41 weeks) and “post date pregnancy” (41–43 weeks). In the “3rd trimester” the amniotic fluid glycoconjugates contained higher relative amounts of glycans terminated by α2-6-linked sialic acid (p < 0.00002) and by α1-6 innermost fucose (p < 0.000001) than those in the 2nd trimester. In contrast, they showed the lower relative amount of fucose linked α1-3 (p < 0.02). At the perinatal period the relative amount of α2-6-linked sialic acid increased (p < 0.03), and it then decreased during delivery (p < 0.02) to the level found in the “3rd trimester” group. In the post date pregnancy all parameters studied increased. The sialyl- and fucosyl-glycotopes of the amniotic fluid glycoconjugates may play an critical role in growth and tissue remodeling of the foetus, as well as may might reflect maturation of a foetus. Additionally, a determination of the glycotope expressions might be helpful in prenatal diagnosis as predictor factors for well being of mother and child.     

Physicochemical Properties and Dissolution Studies of Dexamethasone Acetate-β-Cyclodextrin Inclusion Complexes Produced by Different Methods

Inclusion complexes between dexamethasone acetate (DMA), a poorly water soluble drug, and β-cyclodextrin (βCD) were obtained to improve the solubility and dissolution rate of this drug. Phase-solubility profile indicated that the solubility of DMA was significantly increased in the presence of βCD (33-fold) and was classified as A_L-type, indicating the 1:1 stoichiometric inclusion complexes. Solid complexes prepared by different methods (kneading, coevaporation, freeze drying) and physical mixture were characterized by differential scanning calorimetry, thermogravimetry, infrared absorption and optical microscopy. Preparation methods influenced the physicochemical properties of the products. The dissolution profiles of solid complexes were determined and compared with those DMA alone and their physical mixture, in three different mediums: simulated gastric fluid (pH 1.2), simulated intestinal fluid (pH 7.4) and distilled water. The dissolution studies showed that in all mediums DMA presented an incomplete dissolution even in four hours. In contrast, the complexes formed presented a higher dissolution rate in simulated gastric fluid (SGF pH 1.2), which indicate that these have different ionization characteristics. According to the results, the freeze–dried and kneaded products exhibited higher dissolution rates than the drug alone, in all the mediums.     

Potassium changes in trained subjects after potassium loading and during restriction of muscular activity and chronic hyperhydration

The objective of this investigation was to determine whether urinary and plasma potassium changes developed during prolonged hypokinesia (HK) (decreased number of km/d) in endurance-trained subjects could be minimized or reversed with a daily intake of fluid and salt supplementation (FSS). The studies were performed on 30 endurance-trained male volunteers aged 23–26 yr with an average peak oxygen uptake of 65 mL/kg min during 364 d of HK. All volunteers were on an average of 13.8 km/d prior to their exposure to HK. All volunteers were randomly divided into three groups: 10 volunteers were placed continuously under an average of 14.0 km/d (control subjects), 10 volunteers were subjected continuously to an average of 2.7 km/d (unsupplemented hypokinetic subjects), and 10 volunteers were submitted continuously to an average of 2.7 km/d, and consumed daily an additional amount of 0.1 g sodium chloride (NaCl)/kg body wt and 30 mL water/kg body wt (supplemented hypokinetic subjects). During the prehypokinetic period of 60 d and during the hypokinetic period of 364 d, potassium loading tests were performed with 1.5–1.7 mEq potassium chloride/kg body wt, and potassium, sodium, and chloride excretion in urine and potassium, sodium, and chloride in plasma were determined. In the unsupplemented hypokinetic volunteers, urinary excretion of electrolytes and concentrations of electrolytes in plasma increased significantly as compared to the control and supplemented hypokinetic groups of volunteers. It was concluded that daily intake of fluid and salt supplementation had a favorable effect on regulation of urinary and plasma potassium changes in trained subjects during prolonged HK.     

Stilbene derivatives inhibit the activity of the inner mitochondrial membrane chloride channels

Ion channels selective for chloride ions are present in all biological membranes, where they regulate the cell volume or membrane potential. Various chloride channels from mitochondrial membranes have been described in recent years. The aim of our study was to characterize the effect of stilbene derivatives on single-chloride channel activity in the inner mitochondrial membrane. The measurements were performed after the reconstitution into a planar lipid bilayer of the inner mitochondrial membranes from rat skeletal muscle (SMM), rat brain (BM) and heart (HM) mitochondria. After incorporation in a symmetric 450/450 mM KCl solution (cis/trans), the chloride channels were recorded with a mean conductance of 155 ± 5 pS (rat skeletal muscle) and 120 ± 16 pS (rat brain). The conductances of the chloride channels from the rat heart mitochondria in 250/50 mM KCl (cis/trans) gradient solutions were within the 70–130 pS range. The chloride channels were inhibited by these two stilbene derivatives: 4,4′-diisothiocyanostilbene-2,2′-disulfonic acid (DIDS) and 4-acetamido-4′-isothiocyanostilbene-2,2′-disulfonic acid (SITS). The skeletal muscle mitochondrial chloride channel was blocked after the addition of 1 mM DIDS or SITS, whereas the brain mitochondrial channel was blocked by 300 μM DIDS or SITS. The chloride channel from the rat heart mitochondria was inhibited by 50–100 μM DIDS. The inhibitory effect of DIDS was irreversible. Our results confirm the presence of chloride channels sensitive to stilbene derivatives in the inner mitochondrial membrane from rat skeletal muscle, brain and heart cells.     

The effects of cadmium on zinc absorption in isolated rat intestinal preparations

The effect of cadmium on zinc absorption was studied using an isolated vascularly and luminally perfused rat intestinal preparation.⁶⁵Zn as well as Zn and Cd (both as the chloride salt) were added to the luminal perfusion medium (LPM) at varying concentrations. Over a 90-min period, the amount of Zn appearing in the vascular perfusion medium (VPM) and that retained by the tissue post-perfusion was estimated. Cd at all levels studied (0.03, 0.10, 1.0, and 10.0 μg/mL) reduced the amount of Zn appearing in the VPM in comparison with control perfusions (no detectable Cd in the LPM) when the initial Zn concentration was 5 μg/mL. Similarly, with an initial Zn concentration of 10 or 20 μg/mL, the amount of Zn appearing in the VPM was reduced when the Cd concentration was 0.1 or 1.0 μg/mL. With these same Zn concentrations, the amount of Zn retained by the tissue was higher when the Cd concentration was 10 μg/mL. These results demonstrate that Cd at low concentrations is capable of reducing Zn appearance in the VPM.     

Investigation of some characteristics of enzymes effecting the process of membrane digestion in the paddlefish <Emphasis Type="Italic">Polyodon spathula</Emphasis>

For the first time, a complex investigation of characters of some enzymes effecting the membrane hydrolysis of food in the paddlefish Polyodon spatula is made. The zone of optimum values of temperature of alkaline phosphatase, maltase, and casein-lytic proteinases of the intestinal mucosa is determined within the range from 50 to 60°C, in α-amylase it is shifted towards lower temperatures—10–30°C. A high thermal stability is noted in all investigated enzymes. The maximum level of activity for α-amylase, alkaline phosphatase, and maltase of the intestinal mucosa in the paddlefish Polyodon spatula is within pH 7–9, 9–10, and 6–8, respectively; for casein-lytic proteinases the optimum is at pH 11. In the paddlefish Polyodon spatula, the interaction of nutrients and enzymes of the intestinal mucosa may cause a significant change in the activity level of the enzymes.     

Ontogeny of the sodium pump in embryos of rockfish of the genusSebastes

Synopsis The purpose of the present investigation was to determine at what stages of embryonic development the sodium pump (Na+,K+-activated ATP phosphohydrolase, EC 3.6.1.3) appears and whether the ontogeny of the sodium pump plays a role in the matrotrophic viviparity ofSebastes. Early larval stages (stages 1–14) had embryonic tissues nearly devoid of Na, K-ATPase activity. After epiboly, tissues from embryos taken from both species,S. schlegeli andS. taczanowskii, had significant levels of enzymatic activity coincident with the appearance of neuronal tissue in the head fold (stages 15–20). Maximal levels of specific activities of the enzyme were reached with the onset of the vascular circulation and the opening of the buccal cavity together with maturation of the midgut and hindgut regions of the intestinal tract (stages 26 and 28). Ouabain, a specific inhibitor of Na, K-ATPase, was used to measure survival of embryos placed in different seawater concentrations under in vitro conditions. The most sensitive stages to external ouabain were 26–30. These findings support the hypothesis that the intestinal tract is functional prior to gestation and could be transporting important nutritive material found in maternal ovarian fluid.