Julieta Villanueva

Intestinal Alkaline Phosphatase of the Fish Cyprinus carpio: Regional Distribution and Membrane Association

The distribution of alkaline phosphatase along the carp intestine and also its association to the enterocyte membrane have been studied in order to correlate them with the morphological features and functional specialization of the different intestine portions. The intes- tine was sectioned in seven segments and from each segment a butanol extract of intestinal alka- line phosphatase (IAP) was prepared. The enzyme activity, when expressed as a function of the mucosa or protein content of each segment showed a clear proximo-distal gradient. In addition, IAP was recovered in the precipitate after centrifugation of the homogenate, along the intestine, indicating that it is membrane associated protein. Also, when brush border membranes (BBM) were isolated, IAP was enriched 10-fold. Butanol extracted IAP from all segments exhibited three bands of activity when separated in PAGE-Triton X-100. The slowest migrating band showed a hydrophobic character as it was retained in a phenyl-Sepharose CL-4B column, whereas the other bands represent hydrophilic IAP found in the flow through of the column. Butanol extracted IAP from BBM rendered only one band with hydrophobic character in PAGE-Triton X-100, which could be converted to hydrophilic forms when incubated with phosphatidyl-inositol phospholipase C. These results clearly demonstrate, that in carp as well as in all other species studied, IAP is anchored to the membrane via a glycosyl-phosphatidylinositol. The high IAP content found in the first segment is consistent with the function of dephosphorylation of nutritional compounds pro- posed in higher vertebrates. The involvement of IAP in other physiological roles such as lipid absorption or protein internalization cannot be ruled out. J. Exp. Zool. 279:347-355, 1997.

Univalent cation activation of fructose 1,6-diphosphatase☆

Abstract Fructose 1,6-diphosphatase from several vertebrate sources has been studied with respect to univalent cation activation. All the enzymes tested showed activation by certain univalent cations, K + or NH 4 + being the best activators. A re-evaluation of some properties of fructose 1,6-diphosphatases in the presence of univalent cation activators showed, as studied with pig kidney, rabbit liver, and rabbit muscle fructose 1,6-diphosphatase, that the Mg 2+ -saturation curves were markedly altered by the presence of 150 m m K + . Not only an increase in K a and V max was observed, but also the sigmoidal nature of the Mg 2+ -saturation curves became evident. AMP inhibition, characteristic of most fructose 1,6-diphosphatases, was not significantly altered by the presence of K + in the case of pig kidney, rabbit liver, and fish ( Raja chilensis ) liver fructose 1,6-diphosphatase. Rabbit muscle fructose 1,6-diphosphatase became more sensitive to AMP inhibition, while in the case of fish ( Genipterus chilensis ) liver fructose 1,6-diphosphatase, inhibition by AMP could only be demonstrated in the presence of the univalent cation activators.

Characterization of the major plasma apoliproteins of the high density lipoprotein in the carp (Cyprinus carpio)

Abstract 1. 1. Carp plasma DHL was isolated by a single chromatographic procedure using Affi-Gel Blue. Neither LDL nor VLDL were detected. 2. 2. Thus, HDL which comprises almost 40% of the total carp plasma proteins, also constitutes the major lipoprotein fraction in this fish, yielding two apolipoproteins: apo A-I and apo A-II with molecular weight of 29,500 and 12,000, respectively. These are present in a one to one molar ratio. 3. 3. Carp apo A-I and apo A-II lack cysteine and tryptophan. Although some similarities in the amino acid composition exist with respect to the same apoproteins from other organisms, antisera raised against carp apo-I cross-reacted only, and very slightly, with its rainbow trout counterpart. 4. 4. No reaction was detected with lamprey and the nothotenidae robalo fish. 5. 5. Four plasmatic apo A-I isoforms were identified. No differences were observed in carps acclimatized to winter and summer.

Prolactin gene expression and changes of prolactin pituitary level during the seasonal acclimatization of the carp

The effect of seasonal acclimatization on the extent of prolactin (PRL) gene expression and on the content of this was studied in summer- and winter-carp (Cyprinus carpio) hormone pituitary glands. PRL content in the rostral pars distalis (RPD) was evaluated by immunocytochemistry using antibodies against a cross-linked synthetic peptide comprising the sequence of 15 amino acids which conform to the primary structure of carp PRL. To assess the level of PRL gene transcription, a 24-mer synthetic oligonucleotide probe whose sequence included nucleotides 2041-2064 located in exon V of the carp PRL gene, was used. Employing in situ hybridization assays, a high expression of PRL mRNA was observed in the RPD of summer-acclimatized carp. A negligible level of transcription was observed in tissue sections of pituitary glands from winter-acclimatized carp. Concurrently, immunodetection of the PRL-producing cells in the RPD revealed that the pituitary hormone level was significantly higher in the warm season-adapted carp.

Interaction of cibacron blue and anilinonaphthalenesulphonate with lipoproteins provides a new means for simple isolation of these plasma proteins

The selective interaction of serum proteins with immobilized Cibacron Blue and the binding properties of the dye anilinonaphthalenesulphonate has been used to separate albumin and lipoproteins by affinity chromatography. The novel binding of anilinonaphthalenesulphonate to lipoproteins from the sera of lamprey, fish and mammals provides a simple procedure for the isolation of these plasma proteins, and permit preparation of specific antisera, tools particularly relevant for evolutionary and clinical studies.

Undetectable apolipoprotein A-I gene expression suggests an unusual mechanism of dietary lipid mobilisation in the intestine of Cyprinus carpio

SUMMARY High density lipoprotein (HDL) has been shown to play an important role in the dietary lipid mobilisation in the carp. In spite of this, previous studies have failed to demonstrate the synthesis of the major protein component of HDL, apolipoprotein A-I (apoA-I), in the proximal intestine of the carp. Therefore, the aim of the present study was to evaluate the expression of apoA-I throughout the entire intestine. Curiously, no transcription of the apoA-I gene could be detected either by northern blot or RT–PCR assays in the intestinal mucosa, in clear contrast with the abundant cytosolic immunoreactive apoA-I detected in almost all intestinal segments, which suggests a different origin for this protein. In addition, the detection of specific, but low affinity, binding sites for apoA-I in the carp intestinal brush-border membranes (BBM), and the strong interaction with BBM, which is highly dependent on temperature, points to an important contribution of membrane lipids in apoA-I binding to the intestinal mucosa. This idea was reinforced by the ability of carp apoA-I to associate with multilamellar phospholipid vesicles.

Oral administration of insulin in winter-acclimatized carp (Cyprinus carpio) induces hepatic ultrastructural changes

1. The intestinal absorption of insulin in carps was assessed examining the transepithelial passage of ingested gold-labeled hormone by electron microscopy. Insulin transfer occurred mainly through the intercellular spaces between the enterocytes. 2. When reaching the lamina propria, the gold-labeled hormone gathered predominantly around the granules of the granular cells, and therefore can enter the circulatory system via the blood capillaries which are found in close contact with these cells. 3. Winter-acclimatized carp were also capable of internalizing the hormone when fed with insulin. 4. Furthermore, the absorbed hormone revealed full activity in regard to the observed changes in the ultrastructure of the liver cells of the treated cold-adapted fish. 5. The fish ingesting the hormone underwent the same type of hepatic ultrastructure reprogramming observed when winter-acclimatized carps are injected intraperitoneally with insulin, i.e. conversion to a phenotype corresponding to hepatocytes from summer-adapted carp. 6. The oral absorption of insulin by winter-acclimatized fish and its effect in reversing the cold-adaptive state might be useful for the fish culturing industry.

In vivo levels of aminoacyl-tRNA species during acclimatization of the carp Cyprinus carpio☆

Abstract 1. 1. The behaviour of the tRNA population during the acclimatization process was studied, examining the intracellular levels of aminoacylated-tRNAs in livers from summer and winter adapted carps ( Cyprinus carpio ). 2. 2. The in vivo content of Val-tRNA, Ala-tRNA and Met-tRNA decreased significantly during the summer season, in which Val was 80%, Ala 47% and Met 54% with respect to the values attained in winter. 3. 3. The half-life for the nonenzymic deacylation showed significant variations for the two populations of aminoacyl-tRNA obtained from summer and winter acclimatized fish.

Horseradish Peroxidase Binding to Intestinal Brush- Border Membranes Of Cyprinus carpio . Identification of a Putative Receptor

Morphologic studies have shown that the classic endocytosis tracer horseradish peroxidase (HRP) is actively internalized by vesicular transport in the carp intestine, suggesting the existence of specific binding sites in the apical membrane of enterocytes. The aim of the present study was to develop an in vitro binding assay using isolated carp intestinal brush‐border membranes (BBM) to demonstrate and characterize these specific HRP binding sites. The results obtained show that HRP binding to BBM exhibits a saturable mode and high affinity (Kd = 22 nM). In addition, HRP binding sites are highly enriched in BBM compared to basolateral membranes. On the other hand, HRP interaction with these sites is apparently of an ionic character because binding increased concomitantly with decreasing NaCl concentrations in the assay, reaching a maximum in the absence of NaCl. Other proteins that are also internalized in carp intestine did not significantly inhibit HRP binding to BBM. A lectin‐type of interaction was discarded because neither manan nor ovoalbumin inhibited HRP binding. Proteinase K treatment of BBM reduced HRP binding by 70%, suggesting a proteic nature for this binding site. Finally, ligand blotting assays showed that HRP binds specifically to a 15.3‐kDa protein. Taken together, these results are consistent with the existence of a functional receptor for HRP in carp intestinal mucosa that could mediate its internalization. J. Cell. Biochem. 80:274–284, 2000.