In situ hybridization of DNA sequences in human metaphase chromosomes visualized by an indirect fluorescent immunocytochemical procedure

In situ hybridization and immunocytochemical procedures are described which allow identification and localization of specific DNA sequences in human chromosomes by fluorescence microscopy. With this method the genes coding for 18S and 28S ribosomal RNA (rRNA) were localized on human metaphase chromosomes by in situ hybridization of 18S or 28S rRNA followed by an immunocytochemical incubation with specific anti-RNA-DNA hybrid antiserum. Visualization of the immunocytochemically localized RNA-DNA hybrids was achieved by indirect immuno-fluorescence. The antiserum against RNA-DNA hybrid molecules was raised in a rabbit injected with poly(rA)-poly(dT). The specificity of the sera was determined using a model system of Sephadex beads to which various nucleic acids had been coupled. To obtain optimal specific fluorescence and very low aspecific background staining, several modifications of the in situ hybridization and the immunocytochemical procedures were investigated. The use of aminoalkylsilane-treated glass slides, removal of unbound fluorochrome molecules from the fluorochromelabelled antibody solutions and application of a proteinase K treatment during the hybridization procedure and the immunocytochemical procedure proved to be essential for optimal results.     

Combined 10pter-->p11 and 18pter-->q11 trisomy in a 7-year-old child.

We report a severely mentally retarded, dysmorphic girl aged 7 years with a 47,XX, +der(18), t(10;18)(p11.2;q11.2)mat. The phenotype of our patient is compared with 6 cases of trisomy 10p and 10 cases of trisomy 18q- from the literature. The short trisomic segment 10pter-10p11 appears to affect more the phenotype than the trisomic segment 18qter-q11.     

In vitro fertilization, development, and implantation after exposure of mature mouse oocytes to visible light.

Mature mouse oocytes were exposed prior to in vitro fertilization to visible light during 1, 2, or 4 hr at an intensity of 4,000 lux. Compared to controls cultured under identical conditions but protected from light, exposed eggs did not show any significant modification of cleavage speed and rate. After transfer of blastocysts obtained in vitro in uteri of pseudopregnant females, the implantation rate and the proportion of normal fetuses were not found to be different in relation to preliminary light exposure of oocytes fertilized and cultured in vitro.     

Different fate in vivo of oxidatively modified low density lipoprotein and acetylated low density lipoprotein in rats. Recognition by various scavenger receptors on Kupffer and endothelial liver cells

Human low density lipoprotein was oxidized (Ox-LDL) by exposure to 5 microM Cu2+ and its fate in vivo was compared to acetylated low density lipoprotein (Ac-LDL). Ox-LDL, when injected into rats, is rapidly removed from the blood circulation by the liver, similarly as Ac-LDL. A separation of rat liver cells into parenchymal, endothelial, and Kupffer cells at 10 min after injection of Ox-LDL or Ac-LDL indicated that the Kupffer cell uptake of Ox-LDL is 6.8-fold higher than for Ac-LDL, leading to Kupffer cells as the main liver site for Ox-LDL uptake. In vitro studies with isolated liver cells indicated that saturable high affinity sites for Ox-LDL were present on both endothelial and Kupffer cells, whereby the capacity of Kupffer cells to degrade Ox-LDL is 6-fold higher than for endothelial cells. Competition studies showed that unlabeled Ox-LDL competed as efficiently (90%) as unlabeled Ac-LDL with the cell association and degradation of 125I-labeled Ac-LDL by endothelial and Kupffer cells. However, unlabeled Ac-LDL competed only partially (20-30%) with the cell association and degradation of 125I-labeled Ox-LDL by Kupffer cells, while unlabeled Ox-LDL or polyinosinic acid competed for 70-80%. It is concluded that the liver contains, in addition to the scavenger (Ac-LDL) receptor which interacts efficiently with both Ac-LDL and Ox-LDL and which is concentrated on endothelial cells, an additional specific Ox-LDL receptor which is highly concentrated on Kupffer cells. In vivo the specific Ox-LDL recognition site on Kupffer cells will form the major protection system against the occurrence of the atherogenic Ox-LDL particles in the blood.     

Purification and characterization of a brain-specific protein kinase C substrate, neurogranin (p17). Identification of a consensus amino acid sequence between neurogranin and neuromodulin (GAP43) that corresponds to the protein kinase C phosphorylation site and the calmodulin-binding domain.

Neurogranin, formerly designated p17 (Baudier, J., Bronner, C., Kligman, D., and Cole, R. D.) (1989) J. Biol. Chem. 264, 1824-1828), a brain-specific in vitro substrate for protein kinase C (PKC), has been purified to homogeneity from bovine forebrain. The purified protein has a molecular mass of 7837.1 +/- 0.5 Da, determined by electrospray mass spectrometry. In the absence of reducing agent, dimers and higher oligomers accumulated. On sodium dodecyl sulfate-polyacrylamide gels the protein monomer migrated abnormally with an apparent molecular mass of 15,000-19,000 Da, depending on the percentage of polyacrylamide. The native protein is blocked at its amino terminus. The majority of the primary amino acid sequence was determined following proteolytic and chemical fragmentation. A comparison of the amino acid sequence of neurogranin with that of the brain-specific PKC substrate neuromodulin, revealed a strikingly conserved amino acid sequence AA(X)KIQA-SFRGH(X)(X)RKK(X)K. The two proteins are not related over the rest of their sequences. Neurogranin was shown to be phosphorylated in hippocampal slices incubated with 32Pi and phorbol esters stimulated neurogranin phosphorylation, suggesting that neurogranin is likely to be an in vivo substrate for PKC. In vitro phosphorylation of neurogranin by PKC produced a shift of the isoelectric point of the protein (pI 5.6) to a more acidic value (pI 5.4). Tryptic digestion of the phosphorylated protein yielded a single phosphopeptide having the sequence IQASFR, where the serine residue is the phosphorylated amino acid. This phosphopeptide is part of the conserved sequence shared with neuromodulin and also corresponds to the PKC phosphorylation site on neuromodulin (Apel, E. D., Byford, M. F., Au, D., Walsh, K. A., and Storm, D. R. (1990) Biochemistry 29, 2330-2335). Evidence was obtained suggesting that neurogranin binds to calmodulin in the absence of Ca2+, a feature that also characterizes neuromodulin. We propose that the amino acid sequence shared by neurogranin and neuromodulin reflects a functional relationship between these two proteins and that the consensus sequence represents a conserved PKC phosphorylation site and a calmodulin binding domain that characterizes a class of brain-specific PKC substrates.     

Covalent structure, disulfide bonding, and identification of reactive surface and active site residues of human prostatic acid phosphatase

The pairing of the half-cysteine residues of human prostatic acid phosphatase was established by proteolytic digestion and analysis of the resulting peptide mixtures by fast atom bombardment mass spectrometry (FAB-MS). An independently derived, full length cDNA clone was used as the basis for the interpretation of the FAB-MS data. The sequence of the native protein is that predicted from the present cDNA sequence, except for the carboxyl-terminal end and some possible post-translational deamidations. Isolated human prostatic acid phosphatase was found to have multiple carboxyl-terminal ends, terminating in Thr, Glu, and Asp, corresponding to residues 349-351 of the 354-residue protein that is predicted from the cDNA sequence after removal of a leader peptide. The protein contains no free sulfhydryl groups. The identical monomer chains of the dimeric native enzyme are found to contain three disulfide bonds, specifically Cys-129 to Cys-340, Cys-183 to Cys-281, and Cys-315 to Cys-319. In view of the conserved positions of cysteines in the homologous human and rat liver lysosomal acid phosphatases, an identical disulfide bonding pattern may be predicted for those proteins. The location of a potential antigenic site was established by selective labeling of proximate tyrosine residues predicted to be on the surface. A conserved RHGXRXP sequence is present in the prostatic, lysosomal, Escherichia coli, and yeast acid phosphatases and is predicted to be of mechanistic significance. In addition, residue Arg-54 is shown to be an active site residue by reaction of the enzyme with phenylglyoxal. Interestingly, this residue is present in a sequence RXRY (R,H) that is also present in lysosomal phosphatase and in recently described protein tyrosine phosphatases.     

K+ secretion across frog skin. Induction by removal of basolateral Cl-

We examined the development of K+ secretion after removing Cl- from the basolateral surface of isolated skins of Rana temporaria using noise analysis. K+ secretion was defined by the appearance of a Lorentzian component in the power density spectrum (PDS) when Ba2+ was present in the apical bath (0.5 mM). No Lorentzians were observed when tissues were bathed in control, NaCl Ringer solution. Replacement of basolateral Cl- by gluconate, nitrate, or SO4- (0-Clb) yielded Lorentzians with corner frequencies near 25 Hz, and plateau values (So) that were used to estimate the magnitude of K+ secretion through channels in the apical cell membranes of the principal cells. The response was reversible and reproducible. In contrast, removing apical Cl- did not alter the PDS. Reduction of basolateral Cl- to 11.5 mM induced Lorentzians, but with lower values of So. Inhibition of Na+ transport with amiloride or by omitting apical Na+ depressed K+ secretion but did not prevent its appearance in response to 0-Clb. Using microelectrodes, we observed depolarization of the intracellular voltage concomitant with increased resistance of the basolateral membrane after 0-Clb. Basolateral application of Ba2+ to depolarize cells also induced K+ secretion. Because apical conductance and channel density are unchanged after 0-Clb, we conclude that K+ secretion is "induced" simply by an increase of the electrical driving force for K+ exit across this membrane. Repolarization of the apical membrane after 0-Clb eliminated K+ secretion, while further depolarization increased the magnitude of the secretory current. The cell depolarization after 0-Clb is most likely caused directly by a decrease of the basolateral membrane K+ conductance. Ba2(+)-induced Lorentzians also were elicited by basolateral hypertonic solutions but with lower values of So, indicating that cell shrinkage per se could not entirely account for the response to 0-Clb and that the effects of 0-Clb may be partly related to a fall of intracellular Cl-.     

Localization of xanthine oxidoreductase activity using the tissue protectant polyvinyl alcohol and final electron acceptor Tetranitro BT

We have detected xanthine oxidoreductase activity in unfixed cryostat sections of rat and chicken liver, rat duodenum, and bovine mammary gland using the tissue protectant polyvinyl alcohol, the electron carrier 1-methoxyphenazine methosulfate, the final electron acceptor Tetranitro BT, and hypoxanthine as a substrate. Enzyme activity was localized in rat duodenum at lateral membranes and brush borders of enterocytes and in goblet cells and mucus. Hepatocytes in pericentral areas and especially sinusoidal cells showed high activity in rat liver. Xanthine oxidoreductase was also detected in epithelial cells and milk lipid globules of lactating bovine mammary gland, which is known to contain large quantities of the oxidase form of the enzyme. Chicken liver, which contains an inconvertible dehydrogenase form, also showed high activity in sinusoidal cells. Therefore, we conclude that the tetrazolium reaction demonstrates both the dehydrogenase and the oxidase form of xanthine oxidoreductase. Control activity, in the absence of hypoxanthine or in the presence of the competitive inhibitor allopurinol, was low in all tissues studied. Addition of O2 or NAD to the incubation medium did not change the specific reaction in bovine mammary gland or chicken liver, implying that the dehydrogenase and the oxidase form are not dependent on their natural electron acceptors in this tetrazolium salt reaction. We conclude that the present light microscopic method gives specific and precise localization of xanthine oxidoreductase activity in situ.