Biomedical subjects

R J Cenedella

Publications and source records attributed to R J Cenedella.

At least 37 records · Page 2Linked to original sources

PubMed1995

Role of transcription, translation, and protein turnover in controlling the distribution of 3-hydroxy-3-methylglutaryl coenzyme A reductase in the lens.

PURPOSE: To determine the principle site (epithelium or superficial cortex) of gene transcription and mRNA translation for the regulatory enzyme of lens cholesterol biosynthesis, 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR). To evaluate the contribution of waning enzyme synthesis versus enzyme turnover by proteolysis in accounting for the disappearance of HMGR protein from elongated fiber cells. METHODS: Young rats were treated with lovastatin, a drug that increases transcripts of the HMGR gene and translation of HMGR mRNA in lens secondary to inhibiting cholesterol biosynthesis. The relative concentration of HMGR mRNA in lens epithelium and superficial cortex was estimated by a competitive reverse transcriptase-polymerase chain reaction system. Relative HMGR protein levels were estimated by Western blot analysis. Because lovastatin is cleared rapidly from the lens, the half-life of HMGR protein in epithelium and cortex was estimated by following the disappearance of the increased pool of enzyme protein from each compartment with time after halting drug treatment. RESULTS: Between 75% and 90% of the total content of HMGR mRNA and protein in the epithelium and the superficial cortex of control rat lens was located in the cortex. Treatment with lovastatin increased the content of the mRNA in epithelium and cortex by approximately 0.4-fold and HMGR protein content approximately 5-fold. Although the concentration of HMGR mRNA and protein was similarly increased in epithelium and superficial cortex, approximately 85% to 90% of the total increase in mRNA and protein content was located in the cortex because of that area's greater mass. The half-life for the disappearance of the increased pool of HMGR protein from epithelium and cortex was similar at approximately 14 to 17 hours. CONCLUSIONS: The bulk of HMGR gene transcription and mRNA translation apparently is confined to elongating fiber cells. The 10-fold greater increase in enzyme protein than mRNA levels after lovastatin treatment indicates that enzyme concentration in lens is controlled mainly by effects on HMGR mRNA translation or rates of HMGR proteolysis. The observed rapid turnover of enzyme protein in the epithelium and the superficial cortex, if applicable to the deeper cortex and the homeostatic state (absence of drug exposure), suggests that the gradual disappearance of HMGR protein from the lens could be caused by waning of enzyme synthesis rather than to proteolysis in the absence of continuing enzyme synthesis.

Animals↗

PubMed1994

Spatial distribution of 3-hydroxy-3-methylglutaryl coenzyme A reductase messenger RNA in the ocular lens: relationship to cholesterologenesis.

This study probes the regulation of cholesterol biosynthesis in the ocular lens by estimating the concentration and distribution of the messenger RNA for the rate-controlling enzyme for sterol synthesis, 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR). Because the lens is dependent on biosynthesis for cholesterol, HMGR activity is crucial for the life-long growth of this organ. Young rat lenses were serially divided into several fractions by dissolution in an SDS-containing buffer and each fraction was equated to a percent of the lens radius based upon its protein content. HMGR enzyme activity and cholesterol synthesis has been shown to disappear from the lens cortex over a narrow arc of radius due to loss of enzyme protein. Using a published competitive reverse transcriptase-polymerase chain reaction method for amplifying HMGR mRNA (Powell, E. E., and P. A. Kroon. 1992. J. Lipid Res. 33: 609-614), an average of about 46,000 copies of this mRNA was estimated per lens at all rat ages examined (5-day-old to adult). However, copies/microgram total RNA decreased with aging. The distribution of HMGR mRNA across 95-60% of the lens radius was essentially uniform at 2000-3000 copies/mm3 tissue. But the very superficial cortex contained 5- to 7-times this concentration and accounted for about 35% of the total copies/lens. We estimated that cells in this region each contained 1 to 2 copies of message, a value similar to the estimated copy number of HMGR message in human lymphocytes (Powell and Kroon, ibid). This suggests that the translational efficiency and stability of lens HMGR mRNA must be very high.(ABSTRACT TRUNCATED AT 250 WORDS)

Aging↗

PubMed1993

Isolation of a non-sedimenting membrane fraction from the water soluble fraction of bovine lens.

A membrane fraction was isolated from the water soluble fraction of bovine lens by increasing the density of the water soluble fraction with KBr and subjecting it to overnight centrifugation at 100000 g. We have called this fraction, which floats to the top of the mixture upon centrifugation, the non-sedimenting membrane fraction (NSMF). Electron microscopy of the NSMF revealed that it is composed of the expected membrane structures of unit membrane, fiber junction and cytoskeleton. Significantly less of the total membrane of the NSMF was devoted to fiber junction (22.8%) than in the sedimenting membrane fraction (SMF) (41.1%) prepared by sucrose density centrifugation of the water insoluble fraction. The NSMF accounted for about 7-12% of the total bovine lens membrane, and preliminary experiments demonstrated that a similar fraction could be isolated from the water soluble fraction of lenses from rats, rabbits, chickens and humans. The NSMF contained about 0.9 mg total lipid per mg total membrane protein, which was significantly greater than the value obtained for the SMF (0.5 mg total lipid per mg total membrane protein). The greater relative amount of total lipid in the NSMF was due to a significantly greater relative amount of phospholipid in the NSMF which was further reflected by the observation that the cholesterol: phospholipid molar ration of the NSMF (0.58) was significantly less than that of the SMF (0.88). Thus the relative lipid composition of the NSMF was significantly different than that of the SMF. Although the phospholipid content of the NSMF was greater than that of the SMF, the compositions of the phospholipids in the two membrane fractions were similar. The NSMF possessed essentially the same polypeptides (both extrinsic and intrinsic) which were found in the SMF. The NSMF was found to be distributed throughout the lens in a proportionate manner. We conclude that the NSMF may account for most of the lipid which remains in the water soluble fraction of the normal bovine lens after sedimentation of the water insoluble fraction. This membrane fraction substantially differs from the SMF in terms of structure and relative lipid composition. We speculate that the NSMF may represent a specialised domain of the fiber cell plasma membrane which has been previously unrecognized.

Animals↗

PubMed1993

Calcium activated proteolysis and protein modification in the U18666A cataract.

Proteolytic modifications of specific water soluble lens crystallins during U18666A cataract formation in young rats were identified by two dimensional gel electrophoresis and contrasted with those produced by incubating control lens homogenates with calcium. Protein changes which began in clear precataractous lenses at 12 days age included a decrease in 31 and 27 kDa (likely to be beta B1a and beta A3, respectively) crystallin polypeptides, increase in 25 kDa basic polypeptide, appearance of new polypeptide at 30 kDa and modification of alpha A-crystallin. Further modification of both alpha- and beta-crystallins occurred as cataracts formed; they progressed from early to advanced stage within a span of 4 days. During this period polypeptides beta B1a and beta A3 almost completely disappeared and several new components of 23-26 kDa in beta-crystallin region appeared. Extensive modification of alpha A resulted in appearance of new components of less than 20 kDa. Most of the gamma-crystallins disappeared from the water soluble proteins in advanced cataract lenses of 18 day old rats presumably by leaking out of the lens. The water insoluble proteins which accumulated in the cataract were very similar to modified crystallins which appeared in the water soluble fraction. In vitro incubation of normal lens water soluble proteins with calcium duplicated most of the protein changes seen during cataract progression. Immunoblotting studies with antisera to rat alpha- and beta-crystallins revealed the identity of most of the modified water soluble proteins.(ABSTRACT TRUNCATED AT 250 WORDS)

Androstenes↗

PubMed1993

Antagonism of [3H]fatty acid incorporation into vimentin by sodium pyruvate: pitfalls of protein acylation.

In the course of studying possible fatty acid acylation of vimentin by cultured bovine lens epithelial cells, several potential pitfalls of protein-fatty acid acylation were recognized. Even exhaustive delipidation of vimentin with organic solvents failed to remove all noncovalently associated [3H]palmitate and [3H]myristate. Hydroxylamine treatment of vimentin, separated by sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE), failed to remove either palmitate or myristate derived radiolabel. Hydroxylamine treatment did remove palmitate label from a group of lower molecular weight proteins. The myristate radiolabel associated with vimentin recovered after SDS-PAGE and subjected to acid hydrolysis was shown due to incorporated [3H]amino acids, mainly glutamic acid, generated from the fatty acid. Adding excess sodium pyruvate to labeling media has been used by others to reduce the metabolic conversion of fatty acids to amino acids; however, no direct evidence in support of this antagonism was presented. We observed that inclusion of sodium pyruvate at between 5 and 20 mM in the labeling medium produced a dramatic decrease in incorporation of myristic acid radiolabel into vimentin. However, inclusion of even 20 mM pyruvate did not completely antagonize the metabolic conversion of fatty acid label to amino acids. Furthermore, the sodium pyruvate antagonism could be totally obscured if the exposure of X-ray film by fluorography was even slightly prolonged. The results illustrate the danger in assuming that solvent extraction totally delipidates proteins and that adding sodium pyruvate to labeling media prevents the transfer of fatty acid label to amino acids.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗

PubMed1993

High capacity binding of alpha crystallins to various bovine lens membrane preparations.

This study examines the high capacity binding of intact and carboxyl-terminal-truncated alpha A(alpha A) crystallin to two types of lens membrane preparations; membrane stripped of extrinsic protein and some lipid by extraction with urea and alkali and unextracted membrane isolated by centrifugation of total water insoluble protein on a sucrose gradient (native membrane). High capacity binding of alpha A crystallin to the urea-treated membrane was seen once the alpha A substrate concentration reached about 1 mg/ml of media. The membrane bound up to one mg of alpha A per mg of intrinsic protein (MP26) at a concentration of 5 mg alpha A/ml media, binding 5 to 10 times greater than that seen by others at saturation of the high affinity but low capacity binding sites. No apparent differences were seen between high capacity binding of carboxyl terminal-truncated alpha A (by trypsin) and intact alpha A, although each crystalline could antagonize binding of the other. However, once membrane bound, neither crystallin appeared to grossly displace the other. Using the carboxyl terminal-truncated alpha crystallin as a model substrate, native membrane was seen to have a higher capacity to bind the truncated alpha crystallin than urea-extracted membrane and binding was better correlated with the preexisting alpha A content of the native membrane than its MP26 content. An artificial native membrane was prepared by prebinding the truncated alpha A to urea-extracted membrane. This preparation bound more intact alpha A than urea-extracted membrane bearing no prebound crystallin. We conclude that lens native membrane possesses a high capacity to bind alpha crystallins and that this binding could be mediated through protein-protein interactions with alpha crystallin bound in situ to the membrane as extrinsic protein.

Animals↗

PubMed1993

Regional distribution of 3-hydroxy-3-methylglutaryl coenzyme A reductase activity and protein mass in the ocular lens.

We have attempted to map the regional distribution of 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR) enzyme activity and protein mass along the radial axis of the ocular lens. Because lens plasma membrane is extremely rich in cholesterol and the lens must synthesize this cholesterol, the activity of HMGR could be a key factor controlling lens membrane formation. Lenses of young rats were divided into discrete fractions by gradual dissolution in a tergitol-containing buffer; each fraction was then equated to a specific arc of the radius based upon its protein content. Aliquots of each fraction were assayed for HMGR enzyme activity and protein mass. Relative protein mass was quantitatively estimated by Western blotting using a monoclonal antibody to HMGR with immunoreactivity detected by enhanced chemiluminescence. Lens HMGR possessed a molecular mass of about 97 kDa and localized in the cell's insoluble fraction. Peak levels of both HMGR enzyme activity and protein mass were found in the outer 5% of the lens radius; levels of both decreased precipitously from there to the outer 10% radius mark. This distribution paralleled synthesis of the membrane's cholesterol, phospholipid, and intrinsic protein. We conclude that the abrupt cessation of plasma membrane synthesis in the ocular lens could involve loss of HMGR activity over a narrow arc of the lens radius, and that this activity loss is due to disappearance of enzyme protein.

Animals↗

PubMed1993

Apparent coordination of plasma membrane component synthesis in the lens.

PURPOSE: This study explores the order of assembly of the lens fiber cell plasma membrane. Because the lens must synthesize most of its membrane components, our approach was to map directly the spatial distribution of cholesterol, fatty acid (reflecting phospholipid), and the main intrinsic protein, MP26, synthesis in the lens and, thereby, determine the extent to which membrane component synthesis was coordinated during fiber cell elongation. METHODS: Young rat lenses were incubated with either tritiated water as the substrate for cholesterol and fatty acid synthesis or tritiated leucine as the substrate for MP26 synthesis. We developed a simple technique for uniformly dissolving the decapsulated lens into small fractions by incubating the lens with gentle stirring in sodium dodecyl sulfate-containing buffer. Based on the protein content of each fraction and available information on the radial distribution of protein in the young rat lens, each fraction was equated to a specific percentage of the lens radius. Cholesterol was precipitated from each fraction by digitonin; fatty acids were extracted and isolated by thin-layer chromatography. The MP26 was recovered both by immunoprecipitation from each fraction with anti-MP26 polyclonal antibody and from sodium dodecyl sulfate polyacrylamide gel electrophoresis gels of intact crude membrane, which was isolated from lens fractions by dissolving the lens in a urea containing-buffer. RESULTS: The spatial distribution of incorporation of cholesterol, fatty acid, and MP26 was virtually superimposable, with essentially all the incorporation occurring in the outer 10% of the lens radius and peak incorporation occurring in approximately the outer 3-6% of the radius. CONCLUSIONS: These results indicate that the synthesis of lens membrane components is highly coordinated and imply that the plasma membrane accumulates constant proportions of cholesterol, phospholipid, and MP26 throughout the course of fiber cell elongation.

Animals↗

PubMed1992

Cholesterylene, a newly recognized tissue lipid, found at high levels in the cornea.

In the course of measuring the concentration of cholesterol in an opacified dog cornea by gas-chromatography, relatively large amounts of an unidentified non-saponifiable lipid were recognized. When the unknown lipid was subjected to gas chromatographic-mass spectral analysis it displayed a major ion at m/z 368 M+. and was identified as cholesta-3,5-diene, cholesterylene, by computer match with mass spectral-registry data. Cholesterylene was then shown to be present in the corneas of normal dogs, cows and humans, accounting for 20-25% of the total steroid-sterol in dog corneas and 5-10% in cow and human. Cholesterylene, which can be considered as an extremely nonpolar dehydration product of cholesterol, has not previously been recognized in animal tissues. Although the source of corneal cholesterylene is unknown, preliminary results suggest non-enzymatic formation from cholesterol.

Animals↗

PubMed1992

Examination of a lens 'native' plasma membrane fraction and its associated crystallins.

A bovine lens "native" plasma membrane fraction containing its full compliment of extrinsic proteins was prepared by sucrose density centrifugation of the water insoluble fraction. The major membrane fraction was found at the 25/45% sucrose interface. This fraction contained 73% of the total water insoluble phospholipid, 74% of the total water insoluble cholesterol and 58% of the total urea-insoluble protein. Only 9% of the total urea-soluble protein was membrane associated (extrinsic protein), most (75%) was recovered from the pellet. The major intrinsic protein (8 M-urea-insoluble) of the membrane fraction was MIP28, with lesser amounts of MP17. Extrinsic proteins (8 M-urea-soluble) were examined by SDS-PAGE, isoelectric focusing, immunoblotting and amino acid composition analysis. Approximately 70% of the total extrinsic protein appeared to be alpha A-crystallins and modified alpha A-crystallins. About 20% of the extrinsic protein was apparently beta- and gamma-crystallins. The remainder contained presumed cytoskeletal proteins and perhaps other unidentified polypeptides. The native plasma membrane was found distributed throughout the lens with only minor differences in the quantitative composition of the membrane fraction. We have concluded that the native membrane fraction represents the lens plasma membrane with its extrinsic proteins which exist in vivo. These extrinsic proteins appeared to be primarily acidic alpha-crystallin polypeptides with minor amounts of beta- and gamma-crystallins, and presumed cytoskeletal elements. We speculate that these extrinsic proteins may serve as a nucleation site for the association of other water insoluble protein through protein-protein interactions such as those found in the non-membrane associated urea-soluble protein. Together, these interactions may form a structured cytoplasmic matrix important for the maintenance of lens transparency.

Amino Acids↗

PubMed1992

Selective association of crystallins with lens 'native' membrane during dynamic cataractogenesis.

Plasma membrane with its associated extrinsic proteins was isolated from normal and cataractous rat lenses by centrifugation of the total water insoluble fraction from homogenized lenses on a discontinuous sucrose gradient. Membrane, which we call "native" membrane, was recovered mainly from the 25/45% sucrose interface. Development of the experimental U18666A cataract resulted in plasma membrane shifting to higher density (the 50/55% sucrose fraction) and great increases in the urea soluble protein content of the lens. At early stages of cataract development, most of the increased urea soluble protein was membrane associated, presumably as extrinsic protein. With advancing cataract, most of the urea soluble protein appeared in an essentially membrane-free pellet fraction. The urea soluble protein associated with the cataract membrane was shown by combined IEF, SDS-PAGE, Western blotting, amino acid compositional analysis and protein sequence determinations to be mainly composed of modified alpha- and beta-crystallins. Alpha A-crystallin truncated by not more than 27 residues from the carboxyl terminus plus beta b1 crystallin truncated by 49 residues from the amino terminus were conclusively identified. In addition to beta b1, a population of six alpha-crystallin derived polypeptides were specifically enriched in the cataract membrane fraction. Four of these six alpha-crystallins appear to be truncated from their carboxyl terminus, a modification which should have increased their hydrophobicity. The pellet fraction, which accumulated in the lens nucleus as the cataract advanced, was enriched in urea soluble gamma-crystallin derived polypeptides. We suggest that protein insolubilization in this experimental cataract involves the selective and tight association of principally modified alpha-crystallins to the fiber cell plasma membrane.

Amino Acid Sequence↗

PubMed1991

Lipid composition of lens plasma membrane fractions enriched in fiber junctions.

Little is known about the lipid environment of lens fiber junctions, the plasma membrane structure proposed to be responsible for passage of low molecular weight metabolites between adjacent lens fiber cells. Plasma membranes of the ocular lens are especially rich in fiber junctions. The resistance of junctional domains to disruption by detergent or alkali treatment provides the opportunity to isolate a lens plasma membrane fraction enriched in fiber junctions. When examined by electron microscopy, the fiber junction fraction prepared from bovine lenses was enriched with junctional structures by about twofold when compared to total plasma membrane. We compared the protein, phospholipid, and cholesterol concentration of total plasma membrane with fiber junctional membrane from rat and cow lens and from aged normal cataractous human lenses. The principal finding was that junctional membrane contained 20-40% more total lipid than that of the total plasma membrane. This was due to a proportionate increase in the relative content (mg/mg protein) of both phospholipid and cholesterol. Exclusive of one exception (nucleus of bovine lens), the cholesterol/phospholipid molar ratios of the two fractions were similar. In the bovine nucleus, the cholesterol/phospholipid molar ratio was substantially higher in the fiber junctional-enriched membrane fraction than in the total plasma membrane, suggesting a special association of cholesterol with bovine nuclear fiber junctions. The relative lipid compositions of the plasma membrane and fiber junction-enriched fractions from human normal and cataractous lenses were similar, suggesting that human senile cataractogenesis involves changes in the lens plasma membrane more subtle than would be reflected by gross changes in the membrane lipid composition.

Animals↗

PubMed1990

On the composition and origin of the urea-soluble polypeptides of the U18666A cataract.

The composition and origin of the urea soluble polypeptides which accumulate in the U18666A rat-cataract were studied. Chromatography on Sephacryl S-200 in 7.2 M urea separated the USP into 19-20 and 22-26 kDa enriched fractions. The polypeptide composition of these fractions was probed by immunoblotting of IEF and 2-D electrophoresis gels. The cataract USP largely focused at pHs comparable to alpha- and beta-crystallins. Immunoblotting of 2-D gels showed the USP to be composed predominantly of alpha- and beta-derived crystallins; little gamma-polypeptide was detected in the gels. Some of the insoluble alpha-crystallin appeared to be degraded. Changes in the lens WSP which accompanied the increase in USP were also measured. WSP decreased more than USP increased. Decreases in soluble high molecular weight proteins (alpha- plus beta-crystallins) and medium molecular weight proteins (beta-crystallins) were calculated which together could entirely account for the increased USP. An unexpected decrease in the lens soluble low molecular weight proteins (gamma-crystallins) appeared largely due to the selective leakage of gammas from the lens. The protein content of the ocular humors from eyes with cataracts increased 4 fold and contained polypeptides that focused on IEF like gamma-light crystallin and reacted with the gamma-crystallin antiserum. The cause of the protein insolubilization in the U18666A cataract is unknown but could be partially due to increased aggregation of alpha-crystallins secondary to loss of gamma-crystallins from the lens.

Androstenes↗

PubMed1990

Kinetics of corneal epithelium turnover in vivo. Studies of lovastatin.

The authors developed a direct chemical approach for estimating the rate of turnover of the corneal epithelium in vivo. The method was used to examine the effects of lovastatin, a potent inhibitor of cholesterol biosynthesis, on proliferation and turnover of the epithelium. Corneal DNA was labeled by pulse injection (IP) of the rat with 3H-thymidine, and 3H-labeled DNA was recovered from peripheral and central corneas over the next 15 days. Only the epithelium became labeled, and the loss of label by cell desquamation began 3 days after injection. The loss of 3H-DNA from the cornea (peripheral plus central region) followed first-order kinetics. The half-life of the disappearance was about 3 days. The peripheral cornea became more highly labeled than the central cornea and began to lose 3H-DNA before the central cornea. These observations support the possibility of a higher mitotic rate in the peripheral region and the centripetal movement of a population of peripheral epithelial cells in the normal cornea. The half-lives of the disappearance of 3H-DNA from peripheral and central corneas measured between days 5 and 15 postinjection were identical, both at 3 days. Complete turnover of the corneal epithelium would, therefore, require about 2 weeks (4-5 half-lives). Treatment of the rat with lovastatin had no obvious effects upon the proliferation or turnover of the corneal epithelium. Although lovastatin inhibited corneal 3-hydroxy-3-methylglutaryl coenzyme A reductase, the key regulatory enzyme of cholesterol synthesis, the cornea compensated by induction of this enzyme so that there was no net inhibition of cholesterol synthesis in the cornea.

Animals↗

PubMed1990

Palmitoylation of ocular lens membrane proteins.

Covalent attachment of fatty acids to proteins may be a means of anchoring cytoplasmic proteins to the plasma membrane. The possibility that lens membrane proteins can be fatty acid acylated was studied by incubating the lenses of young rats with 9,10-3H-palmitate. The distribution of 3H-palmitate among the lens membrane polypeptides separated by electrophoresis was determined by fluorography and by direct measurement of radiolabel in sliced gels. 3H-palmitate was found to be incorporated into membrane polypeptide fractions of approximately 19, 30, and 35 kD; the 30 kD fraction appeared to be most highly labeled. The principal lens membrane protein, the main intrinsic protein (MIP 26), was not labeled. This incorporation appeared to be due to covalent attachment rather than to noncovalent binding, and was temperature dependent, independent of protein synthesis, and resistant to displacement by beta-mercaptoethanol. Whether the acylation is enzymatic or nonenzymatic is unclear. The identity of the acylated polpeptides is unknown.

Acylation↗

PubMed1989

Cell cycle specific effects of selenium on the lens epithelium studied in vivo by the direct chemical approach.

We attempted to separate S phase from post-S phase effects of selenium upon the lens epithelium by exposing the lens in vivo to selenium either during S phase synthesis of DNA or immediately after completion of DNA synthesis. Incorporation of 3H-thymidine into lens epithelial cell DNA is complete within about 3 hours after intraperitoneally injecting this substrate. Young rats were given selenium either 5 hours before injecting 3H-thymidine (selenium present at the time of labeled-DNA synthesis) or 5 hours after 3H-thymidine (selenium present immediately after completion of S phase). By measuring changes in the distribution of 3H-DNA between the epithelium and lens body for 14 days after injection we estimated migration times and rates of differentiation of the labeled cell population. When selenium was present during DNA synthesis, DNA labeling was decreased by 70%, net migration time (movement from the germinative zone to the equator) was markedly prolonged and the rate of differentiation was slightly accelerated. Selenium had little affect on these parameters if first present immediately after S phase. We conclude that selenium insult to the lens epithelium is largely confined to germinative epithelial cells in S or pre-S phases of the cell cycle.

Animals↗

PubMed1989

Cholesterol biosynthesis by the cornea. Comparison of rates of sterol synthesis with accumulation during early development.

The origin of the cholesterol needed by the cornea for growth and cell turnover was addressed by comparing absolute rates of sterol synthesis with rates of sterol accumulation during early development of the rabbit. Linearity of incorporation of 3H2O and [14C]mevalonate into digitonin-precipitable sterols with time of incubation in vitro and a lack of accumulation of 14C in intermediates of sterol biosynthesis indicated that tritiated water can validly be used to measure rates of sterol synthesis by the cornea. The rate of sterol synthesis per unit weight of rabbit cornea was constant between 14 and 60 days of age at an average 1.03 nmol of 3H of 3H2O incorporated/mg dry cornea per 8 h. Essentially all of the synthesized cholesterol and most of the cholesterol mass was present in corneal epithelium. The cumulative sterol synthesized over the 46-day period studied exceeded the observed rate of cholesterol accumulation by sixfold. Cholesterol synthesized in excess of the growth requirement was likely used to support turnover of the epithelium which was estimated at 9 days. Removal of cholesterol from the cornea by excretion into tear fluid and clearance by high density lipoproteins are also considered.

Animals↗

PubMed1989

Aging and rates of lens-cell differentiation in vivo, measured by a chemical approach.

We describe a direct and comparatively rapid chemical approach for quantitating rates of lens epithelial cell differentiation in vivo and apply it to a study of the basis of the precipitous decrease in the rate of lens growth in the rat between about 1 and 6 weeks of age. Rates of terminal differentiation of epithelial cells into fiber cells were quantitatively described by a first-order-fractional rate constant (k) for loss of 3H-DNA (labeled from injected 3H-thymidine) from the capsule (epithelium) to fiber cells. The rate constant (expressed in days) was calculated using the half-life estimated from semilogarithmic plots of the percent of total lens 3H-DNA measured in the capsule fraction versus time after injection of 3H-thymidine. The rate constant decreased from about k = 0.18 day-1 at 6 days of age to about 0.09 day-1 at 18-20 days of age and changed little thereafter. The decrease in lens growth at very early ages in the rat is at least partially due to decreases in rates of epithelial cell differentiation.

Aging↗