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D Carper

Publications and source records attributed to D Carper.

At least 37 records · Page 2Linked to original sources
PubMed1990

Cloning and expression of human aldose reductase.

The complete amino acid sequence of human retina and muscle aldose reductase was determined by nucleotide analysis of cDNA clones isolated using synthetic oligonucleotide probes based on partial amino acid sequences of purified human psoas muscle aldose reductase. The cDNA sequence differs substantially in the noncoding and coding regions of recently published sequences of this enzyme. The mRNA for aldose reductase was abundantly expressed in HeLa cells, but only scarcely in a neuroblastoma cell line. Recombinant baculovirus containing one of the muscle cDNA clones was constructed and used to infect Spodoptera frugiperda (SF9) cells. A prominent protein with an apparent molecular size of 36 kDa was identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis in the culture medium as well as in the homogenate of SF9 cells after 2 days of infection. Culture medium or the supernatant fraction of cell homogenates containing this protein had high aldose reductase activity which showed characteristics of the reported human enzyme. These findings indicate that the amino acid sequence reported in this paper represents human retina and muscle aldose reductase and that functional human aldose reductase can be expressed in large amounts in a baculovirus expression system. The result should facilitate refined structural analysis and the development of new specific aldose reductase inhibitors for the treatment of diabetic complications.

Aldehyde Reductase↗
PubMed1990

Induction of aldose reductase expression in rat kidney mesangial cells and Chinese hamster ovary cells under hypertonic conditions.

Rat kidney cortex mesangial cells (MES) and Chinese hamster ovary cells (CHO) responded to hypertonicity (600 mosmol/kg) in culture by accumulating sorbitol. The accumulation of sorbitol was due to increased aldose reductase (AR) activity, apparently brought about by increased levels of AR mRNA and protein. The levels of AR mRNA increased approximately 60-fold in MES cells and 30-fold in CHO cells by 24 h in culture media (300 mosmol/kg supplemented with 150 mM NaCl, 600 mosmol/kg total). AR activity also markedly increased (14- to 16-fold above control), but MES took 4 days and CHO 6 days to reach this maximum. Other osmolytes, raffinose and sorbitol (at concentrations of 250 to 300 mM) elicited the same response as that of 150 mM NaCl. These data show that AR expression is induced in MES and CHO cells under hypertonic conditions. Of special interest is the induction of large amounts of AR in rat kidney cortex mesangial cells, a target tissue of diabetes and a site where excessive accumulation of sorbitol is suspected to be a critical factor in diabetic nephropathy.

Aldehyde Reductase↗
PubMed1990

Increase in aldose reductase mRNA in dog lens epithelial cells under hypertonic conditions.

Aldose reductase (AR) mRNA levels increase when dog lens epithelial cells are exposed to hypertonic conditions. Hybridization of mRNA to an AR cDNA, using Northern and slot blots, showed that AR mRNA is elevated at least fourfold when primary dog lens epithelial cells are grown in media (300 mosmol kg-1) supplemented with 150 mM NaCl (600 mosmol kg-1 final). A time course showed an increase in AR mRNA of approximately twofold by 24 hr with a maximum increase of between four- and eightfold by 48 hr. AR mRNA remained elevated for the duration of the experiment, 8 days. The addition of Tolrestat, an inhibitor of aldose reductase, had no effect on the increased level of AR mRNA in these hypertonically stressed cells. Cells grown in media supplemented with 250 mM sorbitol also showed a substantial increase in AR mRNA. These data indicate, as in other cell types, the lens, a target tissue of diabetes, responds to hypersomotic stress with an induction of AR expression and suggests that AR may play a role in intracellular osmotic regulation.

Aldehyde Reductase↗
PubMed1990

In vivo osmoregulation of aldose reductase mRNA, protein, and sorbitol in renal medulla.

Sorbitol accumulates in renal medullary cells by synthesis from glucose in a reaction catalyzed by aldose reductase. Medullary sodium and urea are high and vary with urinary concentration. Sorbitol varies similarly, consistent with its role as a compatible intracellular organic osmolyte. We measured renal medullary sodium, urea, sorbitol, aldose reductase (protein and activity), and aldose reductase mRNA in rats treated to change medullary sodium and urea. In untreated Brattleboro rats all measurements were low and increased after 7 days of treatment with arginine vasopressin. In contrast, when normal rats were water deprived for 3 days, urea increased out of proportion to sodium, and sorbitol, aldose reductase, and aldose reductase mRNA were unchanged. After 2 h of diuresis, normal rats had lower medullary sodium and urea and reduced mRNA and sorbitol; however aldose reductase did not change. These data are consistent with previous results from cultured cells in which altered extracellular sodium, but not urea, leads to rapid changes in aldose reductase mRNA and slow changes (days) in aldose reductase. In addition, acute decreases in extracellular sodium increase leakage of sorbitol from cells. We also confirm previous results showing medullary glycerophosphorylcholine correlates best with urea, whereas the sum of all compatible osmolytes correlates best with sodium.

Aldehyde Reductase↗
PubMed1989

Relative abundance of aldose reductase mRNA in rat lens undergoing development of osmotic cataracts.

Aldose reductase (AR) messenger RNA concentration was determined in normal rat lens and in lens from rats fed a 50% galactose diet over a period of 20 days. The AR mRNA was detected by using a previously described AR cDNA clone. The relative concentration of the AR mRNA was estimated by cpm of 35S-UTP labeled antisense RNA hybridized to dot-blots prepared from cytosols isolated from single lens, decapsulated lens (cortex) and its respective capsule (epithelia). The results demonstrated that the concentration of the AR mRNA in the epithelium doubled over the 20 day period. Correspondingly, an increase in the concentration of the DNA was also observed, suggesting that the increase in epithelial cytosolic mRNA might be partially due to the increase in the number of epithelial cells occurring in lens undergoing cataractogenesis. The increase in AR mRNA in the epithelia was gradual, and it doubled by day 12 on galactose, while the increase in DNA was rapid and reached an optimum level by about day 4. By day 4 the cortex AR mRNA concentration increased, then rapidly decreased to insignificant levels by day 20. Changes in AR mRNA and in DNA following a high influx of galactose in the lens might suggest a heightened gene response to changes in the cellular environment for the lens epithelium.

Aldehyde Reductase↗
PubMed1988

Characterization of mRNA and genes for aldose reductase in rat.

Aldose reductase (AR; E.C. 1. 1. 1. 21) has been implicated in a variety of diabetic complications. To investigate the expression of this enzyme in target tissues susceptible to such complications, mRNA encoding AR was characterized by Northern blot hybridization in various tissues and cultured cell preparations. The size of mRNA for AR (approximately 1500 bases) was in good agreement with the size determined by sequence analysis. A cDNA probe for AR from rat lens hybridized to the same size species of RNA isolated from cultured dog lens epithelial cells, cultured human retinal capillary pericytes (mural cells), and Y 79 human retinoblastoma cells. In rat tissues, a substantial amount of mRNA was expressed not only in lens, but also in retina, sciatic nerve and kidney medulla. AR mRNA seemed to be less abundant in rat skeletal muscle and brain, and was scarcely present in liver. Furthermore, Southern blot analysis of rat genomic DNA indicated that there are multiple sequences related to that for AR, probably indicating the existence of a multi-gene family.

Aldehyde Reductase↗
PubMed1987

Aldose reductase and p-crystallin belong to the same protein superfamily as aldehyde reductase.

Aldose reductase (EC 1.1.1.21) has been implicated in a variety of diabetic complications. Here we present the first primary sequence data for the rat lens enzyme, obtained by amino acid and cDNA analysis. We have found structural similarities with another NADPH-dependent oxidoreductase: human liver aldehyde reductase (EC 1.1.1.2). The identity between these two enzymes is 50%. Both enzymes share approx. 40-50% homology with p-crystallin, a major lens protein present only in the frog, Rana pipiens. We propose that aldose reductase, aldehyde reductase and p-crystallin are members of a superfamily of related proteins.

Alcohol Dehydrogenase↗
PubMed1987

Phase separation in lens cytoplasm is genetically linked to cataract formation in the Philly mouse.

The variation of the phase-separation temperature, Tc, in lenses was studied during the postnatal development of three genetically different mouse strains: Swiss-Webster, Philly, and the (Swiss-Webster x Philly)F1 hybrid. The general behavior of Tc during early postnatal development has two stages: in stage I, Tc increased to a maximum and then, in stage II, Tc decreased. Philly mice are a strain that develops hereditary cataracts about 36 days following birth. In F1 hybrids of Philly and Swiss-Webster mice, cataracts appeared about 49 days following birth, approximately equal to 13 days later in development than in the Philly mice. In the Philly and hybrid mice, stage I and stage II were followed by stage III in which Tc reached a minimum value and then increased toward body temperature. The values of Tc at birth, the slope of the increase during stage I, and the maximum Tc were characteristic for each mouse strain. These results establish that the behavior of the temperature of the phase separation Tc in mouse lens is linked to the genetic strain of the mice and that the value of Tc at birth is an early indicator of lenses that will develop cataracts and lenses that will develop normally.

Animals↗
PubMed1986

Immunocytochemical localization of the 27K beta-crystallin polypeptide in the mouse lens during development using a specific monoclonal antibody: implications for cataract formation in the Philly mouse.

The Philly mouse develops a hereditary cataract about 5 weeks after birth. Although the causative agent is not known, data suggest that there is a correlation between cataract formation and the selective absence of a 27 kilodalton (27K) beta-crystallin lens polypeptide. The ontogeny of the 27K beta-crystallin polypeptide was examined in normal mice in order to evaluate its role in normal development and determine what impact its absence may have on the Philly mouse lens. A monoclonal antibody was used with the PAP method to immunocytochemically localize the 27K polypeptide in lenses of normal mice during development. beta-Crystallins detected with polyclonal antisera were found in differentiated fiber cells throughout the lens. In contrast, the 27K beta-crystallin polypeptide detected with a specific monoclonal antibody was not found in the fiber cells of the inner part of the lens (nucleus), but was specifically localized in the fiber cells of the outer part of the lens called the cortex. The polypeptide was found only in elongating and differentiated fiber cells and not in mitotically active epithelial cells. Although a minor component of the 2-day-old lens, the 27K polypeptide comprised a large portion of the 16-day-old lens including the anterior and posterior poles. These data show that the 27K polypeptide is a minor component of the embryonic lens, but becomes a major contributor to the postnatal lens. The 27K beta-crystallin lens polypeptide is abundant in the fiber cells of the normal postnatal mouse lens. The absence of the 27K polypeptide in the Philly mouse may contribute to the observed failure of fiber cells to differentiate in the Philly mouse after birth or may be deleterious in some other manner to normal lens development. The selective absence of the 27K beta-crystallin polypeptide, a defect which precedes cataract formation in the Philly mouse, is intriguing since it suggests a relationship between this major lens polypeptide and lens clarity.

Animals↗
PubMed1985

Differential synthesis of rat lens proteins during development.

Developmental regulation of crystallin protein synthesis was observed in rat lenses between embryonic day 19 and postnatal day 21. Studies on lenses incubated in [35S]-methionine and on lens messenger RNAs translated in a reticulocyte lysate showed that several new polypeptides were synthesized in the lens beginning approximately 1 week after birth. One new polypeptide which had a molecular weight of 27 000 comigrated with the beta crystallins on SDS-PAGE and became a predominant component in older lenses. By crossed rocket immunoelectrophoresis and isoelectric focusing, synthesis of several native beta crystallins and one gamma crystallin was detected only in the postnatal lens. Many crystallin proteins were synthesized in the embryonic and the postnatal lens and did not change during the time period studied. These data suggest a differential regulation of the crystallin proteins during development. It appears that the lens undergoes a transition from embryonic to adult crystallin expression during the first weeks after birth. Factors such as maturation of the retina may be necessary for this transition.

Aging↗
PubMed1985

Comparison of the lens crystallin proteins from normal, rd, and rds mutant mice utilizing specific monoclonal antibodies.

The lens proteins from three lines of congenic mice which are homozygous for the gene retinal degeneration (rd) or retinal degeneration slow (rds) or carrying the normal alleles (normal) were analyzed by SDS-PAGE and by immunological reactivity to specific monoclonal antibodies. The lens proteins of normal, rd, and rds mice showed a similar developmental pattern between postnatal day 0 and postnatal day 30. The expression of the 25000 molecular weight (MW) beta-crystallin polypeptide which appears postnatally in the normal lens was not affected by retinal abnormalities in the mutant mice. It is concluded that the regulation of the 25000 MW beta-crystallin polypeptide is not dependent upon differentiation or maintenance of the photoreceptor outer segments or continued presence of the photoreceptor cells.

Animals↗
PubMed1984

Production and characterization of a monoclonal antibody to bovine beta-crystallin.

A monoclonal antibody to a bovine lens 27K beta-crystallin polypeptide has been produced from a rat x mouse hybridoma. The antibody reacts with the 27K polypeptide of bovine, mouse, rat, and human lenses, but does not react to the 27K polypeptide of monkey lens nor does it react with any component in the Philly mouse lens which is missing the 27K polypeptide. The antibody does not recognize any of the other major bovine beta-crystallin polypeptides but does recognize a large number of native beta-crystallin proteins. The antigenic specificity and species cross-reactivity of this antibody provides an excellent opportunity to study many aspects of lens development and cataractogenesis.

Animals↗
PubMed1982

Deficiency of functional messenger RNA for a developmentally regulated beta-crystallin polypeptide in a hereditary cataract.

The messenger RNA for a beta-crystallin polypeptide with a molecular size of 27 kilodaltons, first detected 5 to 10 days after birth in the normal mouse lens and the Nakano mouse cataract, was not detected in the Philly mouse cataract with translation in vitro. The heterozygous Philly lens had intermediate levels of the 27-kilodalton beta-crystallin polypeptide and exhibited delayed onset of the cataract. The deficiency of functional 27-kilodalton beta-crystallin messenger RNA is the earliest lesion reported yet for the Philly lens and points to a transcriptional or posttranscriptional developmental defect in this hereditary cataract.

Animals↗