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Biomedical subjects

I Thalmann

Publications and source records attributed to I Thalmann.

At least 37 records · Page 2Linked to original sources
PubMed1994

Protein profile of human perilymph: in search of markers for the diagnosis of perilymph fistula and other inner ear disease.

Recent developments in high-resolution two-dimensional polyacrylamide gel electrophoresis, combined with amino acid sequencing and computer-assisted image analysis, have allowed separation of approximately 100 proteins and identification and quantitation of some 30 proteins in human perilymph. The majority of proteins were found to be present in perilymph at levels in basic agreement with the total protein gradient between perilymph and plasma (1:35). However, several striking differences were observed: (1) beta 2-transferrin, known to be absent from normal plasma but present in cerebrospinal fluid, was detected in perilymph at a concentration roughly equal to that in cerebrospinal fluid; and (2) two high-density lipoprotein-associated apolipoproteins--apo D (formerly PLS:33) and apo J or NA1 and NA2 (formerly PSL:29/30), the latter showing identity with SP40/40, or cytolysis inhibitor--were found to be present at concentrations 1 to 2 orders of magnitude higher when examined in terms of total protein and to be comparable with or higher than plasma levels when examined in terms of absolute concentrations. The functional significance of the extremely high levels of the two apolipoproteins is not known at this time. An attempt was made to use beta 2-transferrin, as well as apo D and apo J (NA1/NA2), as markers for the diagnosis of perilymph fistula, one of the most controversial and challenging problems for the otologist today. It was determined that the technique is indeed applicable when relatively pure fistula samples are analyzed. Limitations and potential improvements of the technique are discussed. In addition, the potential usefulness of two-dimensional polyacrylamide gel electrophoresis in other pathologic conditions of the inner ear is discussed briefly.

Apolipoproteins↗
PubMed1993

Uronic acid-containing glycosaminoglycans and keratan sulfate are present in the tectorial membrane of the inner ear: functional implications.

The tectorial membrane is a gel-like, acellular connective tissue overlying the microscopic organ of Corti--the auditory sensory structure. It is instrumental in the sound-synchronous deflection of the stereocilia of the hair cells, a central event in auditory transduction. It is well established that collagen, primarily type II, constitutes the major protein of the tectorial membrane, with smaller amounts of types IX and XI also present. However, conclusive information on the proteoglycans in this structure is lacking. Tectorial membranes were extracted with a 4 M guanidine--HCl solvent, and proteoglycans isolated after ethanol precipitation and collagenase treatment. A colorimetric assay based on the binding of the cationic dye safranin O to glycosaminoglycans, in combination with enzymatic techniques, detected significant amounts of chondroitin sulfate and keratan sulfate (0.29 and 0.17% on a wet weight basis, respectively). Agarose-polyacrylamide electrophoresis of chondroitinase-digested samples revealed a core protein with a similar molecular mass to that of the large cartilage proteoglycan aggrecan. This proteoglycan reacted with the antibody 3-B-3 (recognizing modified chondroitin 6-sulfate linkage region oligosaccharides). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed several low molecular mass proteins which reacted with 5-D-4, specific for keratan sulfate, one of which showed characteristics of fibromodulin. Comparison of the quantitative aspects of various connective tissue components of tectorial membrane with other type II collagen-containing structures revealed that this tissue resembles highly hydrated cartilage.

Animals↗
PubMed1993

Partial amino acid sequences of organ of Corti proteins OCP1 and OCP2: a progress report.

Progress in amino acid sequencing of two low-molecular weight acidic proteins of unknown function which are present at high concentrations in the organ of Corti is reported. These two proteins, provisionally termed OCP1 and OCP2, were originally demonstrated by two-dimensional polyacrylamide gel electrophoresis; their presence at high concentrations in the inner ear sensory epithelia strongly suggests that OCP1 and OCP2 serve some important function in the ear. In the present paper, extension of the amino-terminal sequence of OCP2 to 71 residues is described. In addition, the first results on sequencing of OCP1 are presented. Computer algorithms are used to predict important structural features, and the sequences are analyzed for probable phosphorylation, glycosylation, and Ca-binding sites. Projected further studies in immunochemistry and molecular biology of OCP1 and OCP2 based on the amino acid sequencing data are discussed.

Amino Acid Sequence↗
PubMed1993

Collagen of accessory structures of organ of Corti.

It was previously demonstrated that about 40% of the protein of the tectorial membrane of the guinea pig consists of collagen type II, with lesser amounts of type IX and XI. In this paper we extend these studies on the tectorial membrane to the basilar membrane and to other accessory structures, the spiral ligament and spiral limbus. Earlier immunohistochemical data indicated that no collagen type II is present in the basilar membrane of the newborn guinea pig, but that it is present in the area of the basilar membrane in the embryo. However, by means of stringent extraction procedures we have determined biochemically that collagen type II and lesser amounts of type XI are present in the basilar membrane of the adult guinea pig, at similar levels (on the basis of total protein) to the tectorial membrane. Levels of collagen type II are much lower in the spiral ligament and spiral limbus. The presented studies demonstrate that classical techniques of collagen chemistry can be applied at the microscale on minute tissue elements. The significance of the presence of collagen in the tectorial membrane and basilar membrane is discussed in the light of known mechanical properties of these structures.

Amino Acids↗
PubMed1993

[Biochemical profile of proteoglycans and glucosaminoglycans of the mammalian tectorial membrane].

The tectorial membrane is an acellular connective tissue which plays an essential role in cochlear function. While a comparatively large amount of information is available on the collagen network of the tectorial membrane, studies on the biochemical nature of this highly hydrated matrix, which is composed of proteoglycans (PGs) and glycosaminoglycans (GAGs), have been quite limited. Previous reports on the biochemical analysis of the tectorial membrane have failed to detect uronic acid, which is present in large amounts in all mammalian GAGs except keratan sulfate. Applying a colorimetric assay based on the binding of GAGs to cationic dye Safranin-0 in combination with enzymatic techniques, we were able to measure GAGs in the murine tectorial membrane. Approximately 0.3% uronic acid-containing GAGs (mainly in the form of chondroitin/dermatan sulfate) and 0.17% keratan sulfate were detected in the tectorial membrane (both on a wet weight basis). In addition, various types of electrophoresis revealed one large PG with a molecular mass similar to that of the large type cartilage PGs and three small PGs, containing chondroitin sulfate and keratan sulfate side chains, respectively. Judging by coelution of standards, one of the small PGs seemed to correspond to fibromodulin, which has at least one keratan sulfate side chain, and binds to type I and type II collagen to regulate collagen organization in tissues. Our results suggest: (1) Donnan equilibrium is established in the tectorial membrane because sulfated GAGs are highly negatively charged and consequently bring about an influx of large amounts of water and cations into the matrix.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1992

Protein profiles of perilymph and endolymph of the guinea pig.

Results of protein separation of guinea pig plasma, perilymph, and endolymph by means of high-resolution two-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis are presented. Several proteins are present in perilymph at levels in basic accord with the total protein gradient with respect to plasma; however, others are present in perilymph at levels comparable to plasma levels, and one protein low molecular weight protein, PLS:33, is eight times higher. In addition, a high molecular weight protein is shown to be present at similar levels in the two compartments. These findings indicate that ultrafiltration cannot be the sole mechanism of perilymph production. Endolymph proteins are uniformly five to eight times lower than perilymph levels, essentially following the total protein concentration gradient between the two compartments. This supports the view that endolymph is derived from perilymph rather than directly from blood.

Animals↗
PubMed1990

Partial amino acid sequence of organ of Corti protein OCP-II.

We have previously described two low-molecular-weight, highly acidic proteins which are present in the organ of Corti in extremely high concentrations. Since the function of these proteins is not known, they have been assigned the tentative names OCP-I and OCP-II. In the hope of obtaining information about their function through homology studies, we have initiated amino acid sequencing of these proteins. We have recently succeeded in obtaining a brief amino-terminal sequence of OCP-II. We now report on a significant extension of the amino-terminal sequence of OCP-II and our first results on sequences of peptide fragments obtained by limited digestion with V8 protease. Together, the sequenced segments account for about one-third of the total sequence. Comparisons with the sequences of known proteins suggest that OCP-II is not a structural protein, but that it may exhibit biologic activity.

Amino Acid Sequence↗
PubMed1990

Biochemical features of major organ of Corti proteins (OCP-I and OCP-II) including partial amino acid sequence.

Further biochemical and biophysical characterization of two low-molecular-weight, strongly acidic proteins that are present at extremely high levels in the organ of Corti, tentatively named OCP-I and OCP-II, is presented. The two proteins are also present, although at much lower levels, in the vestibular end-organs and a variety of other inner ear tissues; they have not been observed in other systems. OCP-I and II are highly soluble and do not contain appreciable amounts of carbohydrate. The two proteins, originally described in the guinea pig, are compared electrophoretically with the corresponding proteins in several other mammalian species. Preliminary data on the amino acid composition of the two proteins are presented. Moreover, the amino-terminal sequence of a 22-residue segment of OCP-II is shown and compared to the sequences of known proteins.

Amino Acid Sequence↗
PubMed1987

Composition and supramolecular organization of the tectorial membrane.

We have shown that collagen accounts for approximately 40% of the total protein of the tectorial membrane (TM) of the guinea pig and have estimated several essential parameters of TM composition including dry weight, wet weight, and water content. The major collagenous protein was definitively identified as type II collagen by SDS-PAGE, CNBr peptide mapping, and immunoblot assays. Quick-freeze, deep-etch electron microscopy of the guinea pig TM demonstrated a dense meshwork of fibers embedded in a complex microfibrillar matrix which may consist of proteoglycans; the larger fibers were similar in size and appearance to type II collagen fibers of elastic cartilage. Finally, the comparative free amino acid profiles of TM strongly suggest that the TM is chemically transparent with respect to endolymph. Thus, the TM appears to consist of a highly hydrated matrix mechanically stabilized by type II collagen fibers.

Amino Acids↗
PubMed1986

Collagen--the predominant protein of the tectorial membrane.

Evidence is presented that, in contrast to the traditional view, a large proportion of the proteins of the tectorial membrane (TM) consists of collagen, primarily type II: characteristic amino acid composition of TM, including high levels of glycine, hydroxyproline and hydroxylysine; comigration of the main TM protein with appropriate collagen standards in two-dimensional gel electrophoresis; digestion of the main TM protein band following treatment with bacterial collagenase; one- and two-dimensional peptide mapping of cyanogen bromide digests of the TM exhibits patterns characteristic for collagen type II.

Amino Acids↗
PubMed1984

Support of cochlear metabolic and ion transport processes solely by perilymphatic perfusion.

Morphologic considerations would seem to suggest that the cochlear duct could not be maintained in a fully functional state in the absence of a blood supply. We found, however, that perilymphatic perfusion could be used as a substitute for the normal vascular circulation. The criteria used to determine cochlear function included (1) normal endocochlear potential, (2) normal net secretory flux of rubidium (as a tracer for K), and (3) normal levels of ATP in both the organ of Corti and the stria vascularis. All criteria were satisfied by our perfusion regimen.

Adenosine Triphosphate↗
PubMed1982

Adenine nucleotides of the organ of Corti under metabolic stress.

The purpose of the reported experiments was to measure the concentrations of adenosine triphosphate (ATP), adenosine diphosphate (ADP), and adenosine monophosphate (AMP) in the organ of Corti in order to arrive at estimates of three commonly used adenylate ratios. Under normal conditions the concentrations of ATP, ADP, and AMP amounted to 15.8, 3.9, and 0.53 mmoles/kg dry weight, respectively. Of the three substances, AMP is the most sensitive indicator of metabolic stress, since ischemia of 65 seconds leads to an increase of 155%. Under normal conditions the adenylate energy charge, the energy status, and the phosphorylation state amounted to 0.83, 4.1, and 2.5 gram wet weight/mumole, respectively. Within 10 minutes of ischemia the energy charge had declined by 26%, the energy status by 50%, and the phosphorylation state by 76%. The apparent equilibrium constant of adenylate kinase of the organ of Corti was found to be 0.55. The potential significance of these data and their relationship to the situation in the stria vascularis are discussed.

Adenosine Diphosphate↗
PubMed1982

Effect of substrate-free vascular perfusion upon cochlear potentials and glycogen of the stria vascularis.

The effect of vascular perfusion of the anterior inferior cerebellar artery with synthetic blood containing no metabolic substrates upon the endolymphatic potential (EP) and the cochlear microphonics (CM) was determined in the guinea pig. In substrate-free perfusion the potentials were maintained for an average of 84 min. Subsequently, the EP declined at an average rate of 1.4 mV/min until a new steady-state level was temporarily established when the potential had dropped to about 30 mV. The decline of the CM appeared to be accounted for largely by the decline of the EP. During substrate-free perfusion prior to the onset of the decline of the potentials, the level of strial glycogen remained unchanged; glycogen decreased significantly only after the potentials had started to decline. When substrate-free vascular perfusion was accompanied by simultaneous substrate-free perilymphatic perfusion, the potentials started to decline immediately. On the basis of these data, we conclude that strial glycogen plays no role in the prolonged maintenance of the EP during substrate-free perfusion; rather, the potential seems to be maintained by entry of glucose (and presumably other substrates) from perilymph into the stria vascularis.

Animals↗
PubMed1982

Minimal concentrations of metabolic substrates capable of supporting cochlear potentials.

The objective of this study was to determine the capability of glucose analogues, as well as lactate and pyruvate, to maintain the endolymphatic potential and the cochlear microphonics. In addition, the minimum concentration at which different substrates (including D-glucose) were able to sustain the potentials ("critical' concentration) was determined. Synthetic blood containing various substrates at different concentrations was perfused via the anterior inferior cerebellar artery. The critical concentration for D-glucose was found to be 15 mg% (0.83 mM). L-Glucose, galactose and fructose were not able to support the potentials at concentrations as high as 200 mg%. On the other hand, mannose was capable of supporting the potentials; however, the critical concentration (50 mg% or 2.8 mM) was substantially higher than that of D-glucose. Both lactate and pyruvate could support the potentials, but the critical concentrations (8.5 mM and 6.5 mM, respectively) were markedly higher than in the case of glucose, even when the difference of carbon equivalents was taken into consideration. The data are discussed in the context of the intermediary metabolism and possible carrier systems of the stria vascularis.

Action Potentials↗
PubMed1982

Amino acid profiles in inner ear fluids and cerebrospinal fluid.

The levels of 19 amino acids in utricular endolymph, vestibular and cochlear perilymph, and cerebrospinal fluid of guinea pigs were determined using gradient elution reverse phase high performance liquid chromatography of the o-phthaldialdehyde-ethanethiol adducts with fluorescence detection. Aspartate and glutamate were significantly higher in endolymph than in perilymph, in agreement with earlier results on cochlear fluids based on enzymatic fluorometric techniques. All other amino acids tested were significantly lower in the endolymph, in most cases by an order of magnitude. Vestibular perilymph and perilymph of scala vestibuli are virtually identical. Amino acid levels were all higher in perilymph of scala vestibuli than in cerebrospinal fluid; two by an order of magnitude. All differences were statistically significant, with the exception of aspartate. Amino acid levels in perilymph of scala tympani were highly variable dependent upon sampling technique, and no definite values are therefore presented. Comparisons with results from other laboratories, technical pitfalls, and possible implications and interpretations of the results are presented.

Amino Acids↗
PubMed1982

Arguments against a mediating role of the adenylate cyclase--cyclic AMP system in the ototoxic action of loop diuretics.

Previous studies in our laboratory have indicated that adenylate cyclase of the stria vascularis is strongly inhibited in vitro by ethacrynic acid and furosemide. In order to test whether the in vitro effects upon the enzyme are also present under in vivo conditions, ethacrynic acid was perfused perilymphatically for 15 min and 20 min at a concentration of 10(-3) M. Cyclic AMP of the stria vascularis was reduced by 27% and 34%, respectively, but ATP also declined significantly, suggesting unspecific effects. When ethacrynic acid was applied intravenously at a dosage of 50 mg/kg, and the endolymphatic potential allowed to decline to -10mV, no significant changes in cyclic AMP and ATP were seen. The absence of effects upon cyclic AMP in the early stage of systemic intoxication with ethacrynic acid is strong evidence against a mediating role of adenylate cyclase in the ototoxic action of ethacrynic acid. When a bolus of 3 x 10(-2) M furosemide was applied intra-arterially the endolymphatic potential declined at the exceedingly rapid rate of about 10 mV/sec, strongly suggesting that the action of the drug takes place in the vicinity of the capillaries of the stria vascularis. In view of the proposition that adenylate cyclase appears to be located primarily at eh luminal aspect of the stria vascularis, this constitutes further evidence against a role of the enzyme in the mediation of the specific ototoxic effects of loop diuretics. Other recent evidence against a mediatory role of the adenylate cyclase--cyclic AMP system is discussed.

Adenosine Triphosphate↗
PubMed1981

Steep gradients of amino acids between cochlear endolymph and perilymph.

Levels of 19 free amino acids in cochlear endolymph and perilymph of scala vestibuli of the guinea pig were determined using reverse phase high performance liquid chromatography (HPLC) of the o-phthaldialdehyde-ethanethiol derivatives with fluorescence detection. Aspartate and glutamate were found to be significantly higher in endolymph than in perilymph, confirming results from another study using a different analytical method. The remaining 17 amino acids were significantly lower in the endolymph, in many cases by an order of magnitude. The data are compared with results on utricular endolymph and vestibular perilymph obtained by HPLC in another study from our laboratory. Possible implications and interpretations of the results are discussed.

Amino Acids↗