Biomedical subjects
R Cornell
Publications and source records attributed to R Cornell.
Sentence/vowel correlation in the evaluation of dysphonia.
Considering the widespread use of the sustained vowel in the evaluation of voice disorders, our objective was to study the degree to which the vowel is representative of voice in continuous speech. Phonatory samples were recorded from 20 normal subjects and 60 patients representing commonly occurring voice problems (nodules, unilateral paralysis, and functional). The phonatory samples were evaluated by 25 listeners on the basis of 12 bipolar, semantic differential scales. Factor analysis produced two perceptual dimensions with similar loadings for both vowels and sentences. A correlation of 0.78 was obtained between vowels and sentences on factor 1 scores, "vocal severity," and a correlation of 0.77 was obtained between vowels and sentences on factor 2 scores, "pitch/quality." Six of the 80 pairs of sentences and vowels differed by two scale points or more on vocal severity. Results suggest that although a relatively strong relationship exists between the two measures, sustained vowel sounds may not be an adequate clinical index to the dysphonic severity of continuous speech.
Acoustic prediction of severity in commonly occurring voice problems.
The relative effectiveness of three acoustic measures (jitter, shimmer, and harmonic/noise ratio) in predicting the dysphonic severity of a diverse clinical population singly and together was investigated. Phonatory samples were recorded from 20 normal subjects and 60 patients representing 3 laryngeal groups (nodules, paralysis, and functional). The phonatory samples were evaluated by 22 listeners using a 7-point equal-appearing interval scale. Shimmer produced a bivariate correlation of 0.54 with dysphonic severity; harmonic/noise ratio correlated -0.32 with dysphonic severity; and jitter produced no significant correlation with severity. The combination of acoustic variables through multiple regression analysis produced a correlation of 0.56, with only shimmer and average F0 contributing to the correlation. For this particular clinical population, therefore, findings indicated that (a) none of the variables was strongly correlated with dysphonia ratings, and (b) a combination of acoustic predictors was no more successful than a single predictor of dysphonic severity, namely, shimmer.
Acoustic correlates of pathologic voice types.
Listeners classified 49 samples of vowels /a/ and /i/ on the basis of four voice types: hoarse, breathy, strained, and normal. The vowels were analyzed acoustically for mean harmonic/noise differences in four spectral regions, average fundamental frequency, natural logarithm of fundamental frequency, and jitter. Discriminant analysis showed that classifications of voice type were made with 80% accuracy using three acoustic parameters: (a) mean harmonic/noise difference factor (1-3.5 kHz), (b) natural log of fundamental frequency, and (c) vowel type. The significance of these particular acoustic parameters for the perception and classification of voice types is discussed.
Chemical cross-linking reveals a dimeric structure for CTP:phosphocholine cytidylyltransferase.
CTP:phosphacholine cytidylyltransferase (EC 2.7.7.15) was purified from rat liver according to the method of Weinhold et al. (Weinhold, P. A., Rounsifer, M. E., and Feldman, D. A. (1986) J. Biol. Chem. 261, 5104-5110). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis with or without beta-mercaptoethanol revealed a single major band of 42,000 daltons. This band corresponds to the 45-kDa catalytic subunit isolated by Feldman and Weinhold (Feldman, D. A., and Weinhold, P. A. (1987) J. Biol. Chem. 262, 9075-9081). A minor component of 84,000 daltons was intensified in nonreducing gels when the sulfhydryl reducing agent, dithiothreitol, was removed from the enzyme preparation by dialysis. Reduction with dithiothreitol and electrophoresis in the second dimension showed that this 84-kDa protein was derived from the 42-kDa protein. This result suggested that the 42 kDa protein can be converted to an 84-kDa protein by disulfide bond formation. Reaction with the thiol-cleavable cross-linking reagents, dithiobis(succimidyl propionate) or dimethyl-3,3'-dithiobispropionimidate, converted the 42-kDa cytidylyltransferase subunit into a diffuse band approximately twice its molecular mass. Disulfide reduction and electrophoresis in the second dimension showed that this band was derived exclusively from the 42-kDa subunit. This cross-linking pattern was observed when cytidylyltransferase was bound to a Triton X-100 micelle or when bound to a membrane vesicle containing phosphatidylcholine, oleic acid, and Triton X-100. Reaction of the fully reduced enzyme with glutaraldehyde also generated a cross-linked dimer. All three cross-linking reagents inactivated the enzyme. Reduction of the disulfide cross-linkers with dithiothreitol partially reactivated the transferase. When Triton was removed from the enzyme preparation by DEAE-Sepharose chromatography, reaction of the detergent-depleted enzyme with glutaraldehyde generated a band corresponding to a hexamer and higher molecular weight aggregates. The dimeric form was regenerated by addition of either Triton X-100 or phosphatidylcholine-oleic acid vesicles. We conclude that the purified, native cytidylyltransferase, when bound to a detergent micelle or membrane vesicle, is a dimer composed of two noncovalently linked 42-kDa subunits. In the absence of a membrane or micelle, the dimers self-aggregate in a reversible manner.
Translocation of CTP: phosphocholine cytidylyltransferase from cytosol to membranes in HeLa cells: stimulation by fatty acid, fatty alcohol, mono- and diacylglycerol.
Addition of oleate, oleyl alcohol, or palmitate to HeLa cell medium resulted in a rapid stimulation of PC synthesis and activation of CTP: phosphocholine cytidylyltransferase. Stimulation was optimal with 0.35 mM oleate, 0.3 mM oleyl alcohol and 5 mM palmitate, or 1 mM palmitate if EGTA were added to the medium. The cytidylyltransferase was activated by translocation of the inactive cytosolic form to membranes. In untreated cells approx. 30% of the total cytidylyltransferase was membrane bound, while in treated cells, 80-90% was membrane associated. Addition of bovine serum albumin (10 mg/ml) to cells previously treated with oleate (0.35 mM) rapidly removed cellular fatty acid, and the membrane-bound cytidylyltransferase activity returned to approx. 30%. Similar results were obtained by extraction of membranes with albumin in vitro. Although 95% of the free fatty acid was extracted, 30-40% of the membrane cytidylyltransferase remained bound. Translocation of cytidylyltransferase between isolated cytosol and microsomal fractions was promoted by addition of oleate, palmitate, oleyl alcohol, and monoolein. Addition of diacylglycerol, lysophosphatidylcholine, lysophosphatidylethanolamine, calcium palmitate, and detergents such as Triton X-100, cholate or Zwittergent did not stimulate translocation of the enzyme. Addition of oleoyl-CoA promoited translocation, however, 40% of it was hydrolyzed releasing free oleic acid. Cytosolic cytidylyltransferase bound to microsomes pre-treated with phospholipase C, which had 7-fold elevated diacylglycerol content. Fatty acid-promoted translocation was blocked by Triton X-100, but not by 1 M KCl. These results suggest that a variety of compounds with differing head group size and charge, and number of hydrocarbon chains can function as translocators, and that hydrophobic rather than ionic interactions mediate the binding of cytidylyltransferase to membranes.
Binding of CTP: phosphocholine cytidylyltransferase to large unilamellar vesicles.
We have studied the binding of CTP: phosphocholine cytidylyltransferase from HeLa cell cytosol to large unilamellar vesicles of egg phosphatidylcholine (PC) or HeLa cell phospholipids that contain various amounts of oleic acid. A fatty acid/phospholipid molar ratio exceeding 10% was required for CTP: phosphocholine cytidylyltransferase binding to liposomes. At a fatty acid/phospholipid molar ratio of 1; 85% of the cytosolic CTP: phosphocholine cytidylyltransferase was bound. The enzyme also bound to liposomes with at least 20 mol% palmitic acid, monoolein, diolein or oleoylacetylglycerol. Oleoyl-CoA did not promote enzyme binding to liposomes. Binding to oleate-PC vesicles was blocked by Triton X-100 but not by 1 M KCl, and was reversed by incubation of the vesicles with bovine serum albumin. Cytidylyltransferase bound to egg PC vesicles that contained 33 mol% oleic acid equally well at 4 degrees C and 37 degrees C. The enzyme also bound to dimyristoyl- and dipalmitoylphosphatidylcholine vesicles containing oleic acid at temperatures below the phase transition for these liposomes. Binding of the cytidylyltransferase to egg PC vesicles containing oleic acid, monoolein, oleoylacetylglycerol or diolein resulted in enzyme activation, as did binding to dipalmitoylPC-oleic acid vesicles. However, binding to egg PC-palmitic acid vesicles did not fully activate the transferase. Various mechanisms for cytidylyltransferase interaction with membranes are discussed.
The supply of both CDP-choline and diacylglycerol can regulate the rate of phosphatidylcholine synthesis in HeLa cells.
The incorporation of [methyl-14C]CDP-choline into phosphatidylcholine was measured in HeLa cells permeabilized with 0.125 mg digitonin/mL. The rate of phosphatidylcholine formation was influenced by the concentration of CDP-choline in the medium. The CDP-choline:1,2-diacylglycerol cholinephosphotransferase in permeabilized cells showed a Km of 88 microM for CDP-choline. A similar Km value of 104 microM was found for cholinephosphotransferase in microsomes isolated from HeLa cells when assayed in the presence of 2.4 mM dioleoylglycerol. In the absence of added diacylglycerol, the Km for CDP-choline for the microsomal cholinephosphotransferase was only 38 microM. The incorporation of [methyl-14C]CDP-choline into phosphatidylcholine was stimulated by the supply of diacylglycerol in both HeLa cells and isolated microsomes. A 2.4 mM dioleoylglycerol suspension increased cholinephosphotransferase activity fourfold in microsomes. The digitonin-treated cells were impermeable to the dioleoylglycerol suspension. Incubation of permeabilized cells with 150 microM acyl-CoA and 0.8 mM glycero-3-phosphate tripled cellular diacylglycerol levels, causing a doubling in the rate of phosphatidylcholine synthesis. A similar incubation of microsomes with acyl-CoA stimulated phosphatidylcholine synthesis twofold. Furthermore, incubation of microsomes with [3H]diacylglycerol and [14C]CDP-choline showed that both of the substrates were incorporated into phosphatidylcholine at the same rate. This result suggests that the stimulatory effects on cholinephosphotransferase arise from increases in the availability of substrates rather than activation of the enzyme. These results suggest that both in the permeabilized cells and in isolated membranes, the biosynthesis of phosphatidylcholine can be limited by both CDP-choline and diacylglycerol.
Solubilization and reconstitution of cholinephosphotransferase from sarcoplasmic reticulum: stabilization of solubilized enzyme by diacylglycerol and glycerol.
Cholinephosphotransferase (CDPcholine: 1,2-diacylglycerol cholinephosphotransferase, EC 2.7.8.2), which catalyzes the terminal step in phosphatidylcholine synthesis via the CDPcholine pathway, is present in sarcoplasmic reticulum from rabbit skeletal muscle (Cornell, R. and MacLennan, D.H. (1985) Biochim. Biophys. Acta 835, 567-576). The conditions for solubilization and reconstitution of this enzyme were investigated as a preliminary step towards its eventual purification. The activity was not released by treatment of membranes with 1 M KCl, but was solubilized after dissolution of membranes with detergents. Cholinephosphotransferase was inactivated by cholate, deoxycholate, Triton X-100, octylglucoside, Tween-20 or SDS at concentrations which solubilize the membrane. However, the activity could be fully recovered after reconstituting the membrane by adding excess lipid (soybean) and removing detergent by gel filtration, dialysis or by absorption to Bio-Beads. When the membrane was solubilized with octylglucoside or cholate at weight ratios of detergent: membrane protein of at least 10, the activity was irreversibly lost unless stabilizers were added with detergent. The substrate diacylglycerol and glycerol were effective stabilizers.
The capacity of the sarcoplasmic reticulum for phospholipid synthesis: a developmental study.
The activities of three enzymes involved in phospholipid synthesis, sn-glycerol-3-phosphate acyltransferase (EC 2.3.1.15), cholinephosphate cytidylyltransferase (EC 2.7.7.15), and cholinephosphotransferase (EC 2.7.8.2), were assayed in adult skeletal muscle. The acyltransferase and cholinephosphotransferase were concentrated in the sarcoplasmic reticulum, where their specific activities were 80 and 33%, respectively, of the specific activity in liver microsomes. Cytidylyltransferase activity was distributed throughout the cell with most of the activity in the cytosol. Its activity in muscle was only 10% of liver activity. Functional sarcoplasmic reticulum was isolated by density gradient centrifugation after calcium loading in the presence of phosphate. The specific activities of these enzymes wee undiminished in the calcium-loaded fraction, suggesting that these enzymes are intrinsic components of the sarcoplasmic reticulum. In developing muscle (2 and 6 days postnatal) acyltransferase and cholinephosphotransferase activities were also present in a calcium-loaded microsomal subfraction at the same level as in the adult. Cytidylyltransferase activity, on the other hand, was 8-fold higher in developing muscle. In addition, developing muscle had a 3-fold increase in the proportion of cytidylyltransferase associated with the microsomal fraction. These data suggest that sarcoplasmic reticulum has the capacity for phospholipid synthesis in mature and developing muscle, and that the rate of phosphatidylcholine synthesis may be regulated by the levels of cytidylyltransferase and by translocation of this enzyme between the sarcoplasmic reticulum and the cytosol.
Independent synthesis of phospholipid and the intrinsic proteins of the sarcoplasmic reticulum.
The relationship between the synthesis of phospholipids and the intrinsic proteins of the sarcoplasmic reticulum was investigated in differentiating L6 cells in culture. The rates of lipid synthesis and turnover in L6 showed no large variations over the course of differentiation from myoblasts to myotubes while the rate of synthesis of the sarcoplasmic reticulum Ca2+-ATPase steadily increased. Removal of choline from the culture medium after the onset of fusion resulted in a 2-fold inhibition of phosphatidylcholine (PC) synthesis and a 40-50% reduction in total cellular PC content within 36 h. The synthesis and content of phosphatidylethanolamine also declined subsequent to the effect on PC. The amount of newly synthesized phospholipid in the microsomal fraction also decreased 50% in choline-deprived cells. Choline deprivation of myotubes for up to 4 days had no effect on the rates of synthesis of the Ca2+-ATPase or two intrinsic glycoproteins of 53,000 and 160,000 daltons. The newly synthesized proteins were incorporated into PC-deficient microsomal membranes. The synthesis of total cellular protein and total membrane protein was not altered, thus phospholipid:protein ratios declined 2-fold. These observations suggest that the assembly of the sarcoplasmic reticulum is not tightly coordinated with the rate of phospholipid synthesis.
Interrelated lipid alterations and their influence on the proliferation and fusion of cultured myogenic cells.
We have cultured myogenic cells derived from primary explants and a cell line (L6) in a lipid-depleted medium (LDM) and produced large alterations of the fatty acyl and polar headgroup composition and of the cellular sterol levels. These alterations were produced by altering the composition of the media as follows: removing biotin and providing exogenous fatty acid; removing choline and providing exogenous ethanolamine or choline analogues; and by adding 25-OH cholesterol, an inhibitor of 3-hydroxy-3-methylglutarate (HMG)-CoA reductase. Relatively small, secondary alterations of other lipid classes accompany the large primary alteration. In general, they are not obviously compensatory for the primary alteration by retaining some physical property. We have explored the influence of these lipid alterations on myoblast proliferation and fusion into myotubes. In general, considerable variability appears tolerated, but there also appear to be limits. Long-term cultures grown in media containing a single fatty acid do not proliferate indefinitely, and the fatty acid does not become the sole fatty acyl component of the phospholipids. This phenomenon is also observed for cultures enriched in phosphatidylethanolamine (PE) or phosphatidyldimethylethanolamine (PDME). The influence of the lipid alterations on fusion is particularly interesting. The inclusion of 25-OH cholesterol inhibits fusion. Enrichment of the fatty acyl chains with elaidate or the polar headgroups with PE also inhibits fusion, but in contrast to that by 25-OH cholesterol, a significant fraction of the myoblasts are aligned and interacting with each other. Oleate enrichment enhances the rate of fusion.
Lipid requirement for cell cycling. The effect of selective inhibition of lipid synthesis.
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Purification of carboxypeptidase G-1 by immunoadsorption.
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Macromolecular absorption. Mechanism of horseradish peroxidase uptake and transport in adult and neonatal rat intestine.
The immature small intestine of neonatal mammals is permeable to gamma globulins as a source of passive immunity. Allegedly, macromolecular absorption ceases when the epithelial cell membrane matures. However, some evidence exists that adult animals retain a limited capacity to transport antigenic and biologically active quantities of large molecules. In this study, the mechanism of absorption of the tracer protein, horseradish peroxidase (HRP), was tested in neonatal and adult rat gut sacs. Transport into serosal fluid was quantitated by enzymatic assay and monitored morphologically by histochemical techniques. A greater transport of HRP was noted in the adult jejunum compared to adult ileum and neonatal intestine. Morphologically, the uptake mechanism in adult intestine was similar to the endocytosis previously reported in neonatal animals Like other endocytotic processes, HRP uptake in adult rats is an energy-dependent process as determined by metabolic inhibitors and temperature-controlled studies. An understanding of the mechanism whereby macromolecules are bound to intestinal membranes and engulfed by them is necessary before the action of physiologic macromolecules such as enterotoxins can be appreciated.
Implications of subacute hepatic necrosis.
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Amygdalectomy and social behavior in the caged stump-tailed macaque (Macaca speciosa).
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