A trematode from the round window of an Atlantic Bottlenosed dolphin's ear.

A trematode from the family Nasitrematidae Yamaguti 1951 was found adhered to the round window in the inner ear of a Bottlenosed Dolphin (Tursiops truncatus). The possibility that parasites could be responsible for changes in acoustic behavior and hearing loss is discussed.     

Large Alterations in Ganglioside and Neutral Glycosphingolipid Patterns in Brains from Cases with Infantile Neuronal Ceroid Lipofuscinosis/Polyunsaturated Fatty Acid Lipidosis

Lipid composition was studied on cerebral tissue from nine children who had died of a progressive encephalopathy called the infantile form of neuronal ceroid lipofuscinosis (INCL) or polyunsaturated fatty acid lipidosis (PFAL). In the terminal stage of the disease, the concentrations of all lipid classes were found to be significantly reduced in the cerebral and cerebellar cortex and white matter. The concentration of gangliosides of the cerebral cortex was 15% and that of cerebrosides (galactosylceramide) in white matter 0.2-5% of the normal values for the children's ages. The reduction of gangliosides mainly affected those of the gangliotetraose series, particularly GD1a. The fatty acids of the linolenic acid series were strongly reduced in ethanolamine and serine phosphoglycerides. A very large increase up to 100-fold of oligoglycosphingolipids of the globo series and two fucose-containing lipids of the neolacto series was found in the forebrain of the three advanced cases examined. The brain tissue also contained very high concentrations of mono-, di-, and trisialogangliosides of the lacto and neolacto series, gangliosides with type 1 chain dominating. The structures of the gangliosides were tentatively identified by gas chromatography-mass spectrometry and monoclonal antibodies with carefully determined epitope specificity. The gangliosides and neutral glycosphingolipids had very similar fatty acid composition, consisting of about 40% stearic acid and 40% C24-acids.     

Structural studies on H+,K+-ATPase: determination of the NH2-terminal amino acid sequence and immunological cross-reactivity with Na+,K+-ATPase

The NH2-terminal amino acid sequence of the 100 kilodalton subunit of porcine gastric H+,K+-ATPase has been determined to be YKAENYELYQVELGPGP. Although the NH2-terminal region of this protein is not similar to the same region of the lamb kidney Na+,K+-ATPase catalytic subunit, other regions of these ATPase proteins appear to be homologous. Both monoclonal and polyclonal antibodies raised to lamb kidney Na+,K+-ATPase and its alpha, but not beta, subunit cross-react with the 100 kilodalton protein of H+,K+-ATPase.     

Identification of an essential sulfhydryl group in the ouabain binding site of (Na,K)-ATPase

Ellman's reagent 5,5'-dithiobis-(2-nitrobenzoic acid) inhibits sodium- and potassium-stimulated ATPase, p-nitrophenyl phosphatase activity, and [3H]ouabain binding to lamb kidney (Na,K)-ATPase. The inactivation of [3H]ouabain binding follows pseudo-first order reaction kinetics at pH values less than or equal to 8.2. The inactivation of [3H]ouabain binding, but not of enzymatic activity, can be blocked by preincubation with ouabagenin, a rapidly reversible aglycone derivative of ouabain. The reduction in [3H]ouabain binding is due to a decrease in the number of binding sites rather than an alteration of the affinity of the enzyme for ouabain. Differential labeling at pH 8.2 with 1.0 mM 5,5'-dithiobis-(2-nitrobenzoic acid), preincubated with or without 5 microM ouabagenin, followed by tryptic digestion and reverse-phase high performance liquid chromatography of the generated soluble peptides reveals a single peptide labeled by the sulfhydryl probe that is protected by ouabagenin. From these results it is concluded that there is a single sulfhydryl group, essential for ouabain binding, presumably located in the ouabain binding site of lamb kidney (Na,K)-ATPase.     

Fluorescence quenching of human orosomucoid. Accessibility to drugs and small quenching agents.

The fluorescence behaviour of human orosomucoid was investigated. The intrinsic fluorescence was more accessible to acrylamide than to the slightly larger succinimide, indicating limited accessibility to part of the tryptophan population. Although I- showed almost no quenching, that of Cs+ was enhanced, and suggested a region of negative charge proximal to an emitting tryptophan residue. Removal of more than 90% of sialic acid from the glycan chains led to no change in the Cs+, I-, succinimide or acrylamide quenching, indicating that the negatively charged region originates with the protein core. Quenching as a function of pH and temperature supported this view. The binding of chlorpromazine monitored by fluorescence quenching, in the presence and in the absence of the small quenching probes (above), led to a model of its binding domain on orosomucoid that includes two tryptophan residues relatively shielded from the bulk solvent, with the third tryptophan residue being on the periphery of the domain, or affected allotopically and near the negatively charged field.     

Cell poking. Determination of the elastic area compressibility modulus of the erythrocyte membrane

Cell poking, a new method for measuring mechanical properties of single cells was used to determine the elastic area compressibility modulus of osmotically swollen human erythrocytes. With this method we determined the force required to indent cells attached to a glass coverslip (Petersen, N.O., W. B. McConnaughey , and E. L. Elson , 1982, Proc. Natl. Acad. Sci. USA, 79:5327. Forces on the order of one millidyne and indentations on the order of one micron were detected. An analysis of these data in terms of a simplified mechanical model yielded the elastic area compressibility modulus. This analysis used a variational approach to minimize the isothermal elastic potential energy density function given by E. A. Evans and R. Skalak (Mechanics and Thermodynamics of Biomembranes, 1980, CRC Press, Boca Raton , FL). Measurements on swollen erythrocytes gave a range of values, depending in part on the osmotic conditions, of 17.9 +/- 8.2 to 34.8 +/- 12.0 mdyn /micron for the elastic area compressibility modulus at 25 degrees C. Fractional area expansion greater than 2.6 +/- 0.8% produced rapid cell lysis. These values were not corrected for the reversible movement of water across the cell membrane in response to hydrostatic pressure gradients. Our results agree reasonably with those obtained by Evans et al. (Evans, E.A., R. Waugh , and L. Melnick , 1976, Biophys. J., 16:585-595.) using micropipette aspiration under similar conditions.     

Determination of three disulfide bonds and one free sulfhydryl in the beta subunit of (Na,K)-ATPase

The oxidation state of the 7 cyst(e)ine residues of the beta subunit of lamb kidney (Na,K)-ATPase was determined. The fluorescent sulfhydryl-reactive reagent 4-(aminosulfonyl)-7-fluoro-2,1,3-benzoxadiazole was utilized before and after reduction of disulfide bonds to determine the number and location of disulfide bonds present in the beta subunit. Treatment of tryptic peptides of the beta subunit separated by reverse phase high performance liquid chromatography with a reducing agent and 4-(aminosulfonyl)-7-fluoro-2,1,3-benzoxadiazole allowed identification of which residues were disulfide-linked. The results indicated that there is only one free sulfhydryl, Cys44, and that Cys125 is disulfide-bonded to Cys148, Cys158 is disulfide-linked to Cys174, and Cys212 is disulfide-bonded to Cys275. All three disulfide bonds were shown to be reduced in the presence of high concentrations of beta-mercaptoethanol and elevated temperature. These conditions also lead to a loss of (Na,K)-ATPase activity.     

Antisense oligonucleotide containing an internal, non-nucleotide-based linker promote site-specific cleavage of RNA.

We have designed and synthesized a series of novel antisense methylphosphonate oligonucleotide (MPO) cleaving agents that promote site-specific cleavage on a complementary RNA target. These MPOs contain a non- nucleotide-based linking moiety near the middle of the sequence in place of one of the nucleotide bases. The region surrounding the unpaired base on the RNA strand (i.e. the one directly opposite the non-nucleotide-linker) is sensitive to hydrolytic cleavage catalyzed by ethylenediamine hydrochloride. Furthermore, the regions of the RNA comprising hydrogen bonded domains are resistant to cleavage compared with single-stranded RNA alone. Several catalytic moieties capable of supporting acid/base hydrolysis were coupled to the non-nucleotide-based linker via simple aqueous coupling chemistries. When tethered to the MPO in this manner these moieties are shown to catalyze site-specific cleavage on the RNA target without any additional catalyst.