Amino acid sequence at the site on protein phosphatase inhibitor-2, phosphorylated by glycogen synthase kinase-3.

The primary structure surrounding the residue on Inhibitor-2 phosphorylated by glycogen synthase kinase-3 has been determined. The sequence is: Lys-Ile-Asp-Glu-Pro-Ser-Thr(P)-Pro-Tyr-His-Ser. This finding will facilitate studies of the effects of hormones on the phosphorylation state of Inhibitor-2 in vivo.

Regulation of epididymal principal cell functions by basal cells: role of transient receptor potential (Trp) proteins and cyclooxygenase-1 (COX-1).

The epithelia lining the epididymides of many species including the human are known to consist of several cell types. Among them, the principal cells are the most abundant and their functions most extensively studied. There are other cell types such as the narrow cells, clear cells, halo cells and basal cells which are scattered along the duct in lesser number. Although these minority cell types have not been studied to the same extent as the principal cells, it is conceivable that their presence are essential to the integrated functions of the epididymis. In the intact epididymis, basal cells can be seen adhering to the basement membrane forming close contact with the principal cells above them. Work in our laboratory has provided evidence that through local formation of prostaglandins, basal cells may regulate electrolyte and water transport by the principal cells. This regulatory process involves two proteins which are exclusively expressed by the basal cells. They are the transient receptor potential (Trp) proteins, which serve as transmembrane pathways for Ca(2+) influx, and cyclooxygenase 1 (COX-1), a key enzyme in the formation of prostaglandins. The role of the two proteins in the integrated functions of the basal cells as humoral regulators of principal cells is discussed.

Performance enhancement using nonlinear preprocessing.

Describes a nonlinear preprocessing method to enhance the output performance of a network. The introduction of the nonlinear preprocessing method redistributes the distributions of input and output vectors, and makes the input and output variables more "orthogonal" that results in facilitating the network optimization. In some of the examples, this nonlinear preprocessing technique enables test set error to be reduced by a magnitude of 98%. Three applications of time-series predictions applied to evaluate the performance of the proposed method are presented.

A neural-based crowd estimation by hybrid global learning algorithm.

A neural-based crowd estimation system for surveillance in complex scenes at underground station platform is presented. Estimation is carried out by extracting a set of significant features from sequences of images. Those feature indexes are modeled by a neural network to estimate the crowd density. The learning phase is based on our proposed hybrid of the least-squares and global search algorithms which are capable of providing the global search characteristic and fast convergence speed. Promising experimental results are obtained in terms of accuracy and real-time response capability to alert operators automatically.

Isolation and characterization of monoclonal antibody directed against bovine alpha s2-casein.

A monoclonal antibody 62-1A of isotype IgM, directed against bovine alpha s2-casein, was isolated and characterized. Monoclonal antibody 62-1A recognized bovine alpha s2-11P- and alpha s2-9P-caseins by indirect solid phase radioimmunoassay and Western blot analysis. Crossreactivity toward native genetic variants (A, B, and C) of alpha s1-casein was similar but lower (approximately 60 to 70%) than that for alpha s2-11P-casein). Little or no crossreactivity was observed for other bovine milk or serum proteins. Antibody affinity for alpha s2-11P-casein was 1.3 x 10(9)/M. As little as 25 ng/ml (.5 ng/well) of alpha s2-11P-casein was detected by solid phase radioimmunoassay.

Distribution of ADPase activity in the lactating rat mammary gland and its possible role in an ATP cycle in the Golgi apparatus.

A Ca2+/Mg(2+)-stimulated ADPase has been found to occur in the lactating rat mammary gland. The enzyme is membrane associated and occurs in mitochondrial, microsomal, and Golgi apparatus fractions. The pH activity curves for the Golgi apparatus and microsomal fractions display two distinct maxima, one at pH 6.3 and one at pH 7.4. Studies with inhibitors and activators indicate that the enzyme is similar to ADPases found in other tissues and is distinct from the uridine nucleoside diphosphatase previously reported in the mammary Golgi apparatus. The occurrence of ADPase in the Golgi apparatus indicates a possible role for this enzyme in the milk secretory process, while the microsomal enzyme could be involved in extracellular activities.

Subcellular and ultrastructural localization of alkaline phosphatase in lactating rat mammary glands.

The subcellular fractions of lactating rat mammary glands were isolated by differential centrifugation. The mean specific activity of alkaline phosphatase in various fractions was in order greatest to least: microsomes, Golgi, mitochondria, nuclei, and cytosol. Alkaline phosphatase was examined cytochemically by transmission electron microscopy. Alkaline phosphatase activity was localized on myoepithelial membranes, basal and possibly lateral membranes of secretory epithelial cells, and endothelial cells. This finding agreed with biochemical data associating this enzyme activity with microsomes. However, intracellular activities could not be detected on Golgi, secretory vesicles, or apical plasma membranes. Saponin uncovered the activity in that portion of the endoplasmic reticulum of secretory cells adjacent to myoepithelial cells. The identify of this enzyme was further confirmed by selective inhibition studies using dithiothreitol and levamisole. Alkaline phosphatase activities were detected biochemically in lipid droplet "membranes" of secretory epithelium and fat globule membranes. Activity decreased with increasing globule size, indicating that milk alkaline phosphatase originates from lipid droplets of secretory epithelium. The predominance of alkaline phosphatase activity in myoepithelial cell plasma membranes suggests that this enzyme could be involved in cell surface reactions related to oxytocin-mediated milk ejection. In secretory epithelium, it was associated with basal and possibly lateral membranes and lipid droplets that lead to the secretion of milk fat.

Effects of mixed-function oxidase inducers and inhibitors on cytochrome P-450 content, gel electrophoresis profiles, and 3-methylindole-induced lung toxicity in goats.

Effects of mixed-function oxidase inducers and inhibitors on cytochrome P-450 content, gel electrophoresis profiles, and 3-methylindole-induced lung injury were studied in goats. In the first experiment, goats were pretreated with phenobarbital (PB), 3-methylindole (3MI), 3-methylcholanthrene (3MC), or SKF 525-A. Liver, lung, and kidney microsomes were used for cytochrome P-450 and gel electrophoresis studies. Although pulmonary cytochrome P-450 was not affected by any of the pretreatments, hepatic cytochrome P-450 significantly increased (P less than 0.05) after PB and 3MC pretreatment. All pretreatment resulted in changes in liver gel profiles in the 43,000 to 68,000 molecular weight region, but only PB and 3MI pretreatment altered gel profiles in the lung. In the second experiment, pretreated goats were infused with 3MI. The relative severity of lung injury in these animals, based on clinical signs, lung lesions, lung weight, and mean survival time was as follows: SKF 525-A pretreated group greater than PB-pretreated group = unpretreated group greater than 3MI-pretreated group greater than or equal to 3MC-pretreated group. The results indicate that changes in 3MI-induced lung injury are accompanied by changes in gel profiles in the molecular weight region of 43,000 to 68,000.

Distribution of adenosine-5'-diphosphatase activity in the lactating bovine mammary gland.

A Ca2+- and Mg(2+)-stimulated adenosine-5'-diphosphatase has been found in lactating bovine mammary glands. The enzyme is associated with membranes of mitochondrial, microsomal, and Golgi apparatus fractions. The pH activity curves for the Golgi apparatus and microsomal fractions display two distinct maxima, one at pH 5.8 and the other between pH 7.4 and 8.4. Studies with activators and inhibitors indicate the enzyme is similar to adenosine-5'-diphosphatase found in other tissues. Its occurrence in the Golgi apparatus fraction indicates a possible role for this enzyme in the milk secretory process, particularly in ATP cycling in vesicles. Its occurrence in the microsomal fraction suggests a role in plasma membrane functioning.

Synergistic effects of cystic fibrosis transmembrane conductance regulator and aquaporin-9 in the rat epididymis.

The cystic fibrosis transmembrane conductance regulator (CFTR) and aquaporin-9 (AQP-9) are present in the luminal membrane of the epididymis, where they play an important role in formation of the epididymal fluid. Evidence is accumulating that CFTR regulates other membrane transport proteins besides functioning as a cAMP-activated chloride channel. We have explored the possible interaction between epididymal CFTR and AQP-9 by cloning them from the rat epididymis and expressing them in Xenopus oocytes. The effects of the expressed proteins on oocyte water permeability were studied by immersing oocytes in a hypo-osmotic solution, and the ensuing water flow was measured using a gravimetric method. The results show that AQP-9 alone caused an increase in oocyte water permeability, which could be further potentiated by CFTR. This potentiation was markedly reduced by phloretin and lonidamine (inhibitors of AQP-9 and CFTR, respectively). The regulation of water permeability by CFTR was also demonstrated in intact rat epididymis luminally perfused with a hypo-osmotic solution. Osmotic water reabsorption across the epididymal tubule was reduced by phloretin and lonidamine. Elevation of intracellular cAMP with 3-isobutyl-1-methylxanthine increased osmotic water permeability, whereas inhibiting protein kinase A with H-89 (N-(2-[p-bromocinnamylamino]ethyl)-5-isoquinoline sulfonamide hydrochloride) reduced it. These results are consistent with a role for CFTR in controlling water permeability in the epididymis in vivo. We conclude that this additional role of CFTR in controlling water permeability may have an impact on the genetic disease cystic fibrosis, in which men with a mutated CFTR gene have abnormal epididymis and infertility.

Sequence and comparative analysis of the mouse 1-megabase region orthologous to the human 11p15 imprinted domain.

A major barrier to conceptual advances in understanding the mechanisms and regulation of imprinting of a genomic region is our relatively poor understanding of the overall organization of genes and of the potentially important cis-acting regulatory sequences that lie in the nonexonic segments that make up 97% of the genome. Interspecies sequence comparison offers an effective approach to identify sequence from conserved functional elements. In this article we describe the successful use of this approach in comparing a approximately 1-Mb imprinted genomic domain on mouse chromosome 7 to its orthologous region on human 11p15.5. Within the region, we identified 112 exons of known genes as well as a novel gene identified uniquely in the mouse region, termed Msuit, that was found to be imprinted. In addition to these coding elements, we identified 33 CpG islands and 49 orthologous nonexonic, nonisland sequences that met our criteria as being conserved, and making up 4.1% of the total sequence. These conserved noncoding sequence elements were generally clustered near imprinted genes and the majority were between Igf2 and H19 or within Kvlqt1. Finally, the location of CpG islands provided evidence that suggested a two-island rule for imprinted genes. This study provides the first global view of the architecture of an entire imprinted domain and provides candidate sequence elements for subsequent functional analyses.

Properties of [Ca2+ + Mg2+]-adenosine triphosphatases in the Golgi apparatus and microsomes of the lactating mammary glands of cows.

The [Ca2+ + Mg2+]-ATPase activity of bovine lactating mammary gland is associated with membranes. This study compares the ATPase activity in microsomal membranes to that of the Golgi apparatus. The enzyme activity in both fractions hydrolyzed Ca(2+)-ATP and Mg(2+)-ATP. The ATPase activities were inhibited by p-chloromercuribenzoate, indicating the involvement of a sulfhydryl group for activity. Although calmodulin had no effect on the ATPase activities of the two fractions, calmodulin antagonists (chlorpromazine, fluphenazine, and trifluoperazine) were inhibitory. Strong inhibitors of the ATPase activities were vanadate, dicyclohexyl-carbodiimide, La3+, and Zn2+. There were some differences in the activities from two membrane fractions. Although both fractions could hydrolyze all of the triphosphonucleotides, cytidine-5'-triphosphate and uridine-5'-triphosphate were poor substrates for the Golgi enzyme. Detergents diminished the activity of the microsomal enzyme to a much greater extent than the ATPase of the Golgi apparatus. Thus, the intact membrane may be more critical to microsomal activity. The role of these enzymes in Ca2+ accumulation in milk is discussed.