Biomedical subjects

D C Beebe

Publications and source records attributed to D C Beebe.

At least 37 records · Page 2Linked to original sources

PubMed1993

Regulation of lens cell growth and polarity by an embryo-specific growth factor and by inhibitors of lens cell proliferation and differentiation.

We used a double-label method, which monitors the rate at which cells enter S-phase of the cell cycle, to identify factors that control the growth of chicken embryo lens epithelial cells in vivo. With this assay, we identified a mitogen for lens epithelial cells in the anterior segment of the embryonic eye. When the anterior chamber was opened briefly, by tearing the cornea or displacing the lens, the growth-promoting activity was lost. None of the purified growth factors tested replaced this growth activity, including EGF, bFGF, PDGF, IGF-1, IGF-2, TGF beta and mixtures of these factors. However, chicken embryo serum or plasma did cause chicken embryo lens epithelial cells to progress through the cell cycle. The activity in serum was destroyed by heat and protease treatment. It was most active in serum from 10-day embryos, decreased with subsequent development and was undetectable from 2 days after hatching through adulthood. When embryo serum or plasma was mixed with vitreous humor or IGF-1, agents that induce lens fiber cell formation, cell elongation was prevented. In contrast to the mitogenic activity in serum, this inhibitor of differentiation was insensitive to trypsin treatment. We also identified an activity in vitreous humor that inhibited the growth-promoting agent in embryo serum. Plasma proteins readily enter the anterior chamber of the eye of chicken embryos.(ABSTRACT TRUNCATED AT 250 WORDS)

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PubMed1993

Identification of receptor tyrosine kinases in the embryonic chicken lens.

Protein phosphorylation plays a critical role in the control of growth and regulation of many eukaryotic cells. Members of the protein tyrosine kinase (PTK) family of peptides function as growth factor receptors and oncoproteins. A common feature of members of the PTK family is a highly conserved intracellular catalytic domain. We analyzed the chicken lens epithelium, which responds to several known growth factors, for the presence of receptor PTK's. Using reverse transcription polymerase chain reaction (rtPCR) and degenerate primers made to conserved regions within kinase domains, we amplified RNA from embryonic day 6 (E6) lens epithelium and sequenced 135 cDNA clones. Sixteen distinct kinase sequences were obtained. Eight of these sequences represented kinase domains of known mammalian growth factor receptors, and six represented intercellular kinases. Two sequences appeared to code for new kinases. The amino acid identity of the chicken homologs ranged from 80-100% when compared to their mammalian counterparts.

Amino Acid Sequence↗

PubMed1992

Changes in cellular dynamics during the development of the ciliary epithelium.

Eye growth and the formation of the ciliary folds during development depend on the maintenance of normal intraocular pressure. In this study we measured labeling index, packing density, height, and volume of the prospective ciliary epithelial cells during the early development of the eye in chicken embryos. Changes in these values were related to the formation of the ciliary folds and to experimental alterations of intraocular pressure. These variables changed independently in the outer, pigmented layer and the inner, non-pigmented epithelial layer of the ciliary epithelium. The labeling index in the pigmented epithelium declined steadily throughout development. Decreases in cell density and increases in cell height resulted in a four-fold increase in calculated average cell volume in the pigmented epithelium during Hamburger-Hamilton stages 29 and 30, the day before the formation of the ciliary folds. In the non-pigmented epithelium, the labeling index increased abruptly at stage 31, the stage at which ciliary folds begin to form. At the same time there was a three-fold increase in average cell volume. When intraocular pressure was reduced by intubating the eye, the formation of ciliary folds was prevented. Intubation also blocked the increase in labeling index and blunted the increase in cell volume that normally occur in the non-pigmented epithelium at the time of fold formation. In the pigmented epithelium, intubation caused a sustained, three- to six-fold increase in cell volume, but had no effect on labeling index. Based on these data, we suggest that increases in cell number and volume just before and during the formation of ciliary folds contribute to the formation of the ciliary folds.

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PubMed1992

Coincident loss of mitochondria and nuclei during lens fiber cell differentiation.

During normal differentiation, lens fiber cells lose their nuclei, mitochondria, and other membrane-bound organelles. In the present study, a slice preparation of the embryonic chicken lens was used with laser scanning confocal microscopy to study the spatial and temporal patterns of organelle breakdown during embryonic development. At all stages examined, mitochondria in lens epithelial cells were present in perinuclear clusters. In contrast, early in development, lens fiber cells contained extremely elongated mitochondria (> 100 microns) that were distributed throughout the cytoplasm and oriented along the long axis of the cells. By the 8th day of embryonic development (E8), the mitochondria in the central fiber cells began to fragment. At the same time, the nuclei in these cells became smaller and more spherical. By E10, mitochondrial staining in the central fibers became punctate. Electron microscopy of this region revealed swollen mitochondria with disrupted cristae. By E12, cells in the central region of the lens lacked mitochondria and nuclei. The loss of nuclei and mitochondria from a given cell was coincident and abrupt (2-4 hr), occurring in a previously unsuspected domain situated about 300 microns from the anterior surface of the lens. A cytoskeletal component, actin, persisted in the central cells indicating that organelle degradation represents a selective process and not simply the global degradation of supramolecular structures. Throughout embryonic development, the organelle-free region grew at approximately the same rate as the lens and, by the time of hatching, had expanded to match the diameter of the pupil.(ABSTRACT TRUNCATED AT 250 WORDS)

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PubMed1992

Ion concentrations, fluxes and electrical properties of the embryonic chicken lens.

The membrane properties of embryonic chicken lenses were characterized using isotopic and electrical techniques. The lenses had a relatively high water content (80%) and large extracellular space (12.5%). Isotopic uptake measurements indicated that the lens cytoplasm contained 118 mM K+ and 26 mM Cl-. A value for intracellular Na+ of 14 mM was obtained using Na(+)-sensitive microelectrodes. A double-exponential model was used to fit the efflux of 86Rb+, 22Na+, 36Cl- and [3H]mannitol (an extracellular space marker) from the lens. When perfused with artificial aqueous humor (AAH) solution, embryonic lenses exhibited membrane potentials of between -20 and -40 mV. The more negative values were generally observed in lenses from older embryos. A ouabain-sensitive component, contributing -7 mV to the membrane potential, was also identified. The relatively depolarized membrane potentials suggested that the lens membranes were only weakly selective for K+ over Na+. To test this further, lenses were perfused with AAH containing varying concentrations of K+. The resulting changes in potential were interpreted in terms of the Goldman model. The best fit of the Goldman potential equation indicated that, in the presence of ouabain, the chicken lens membranes had a relative permeability to K+, Na+ and Cl- of 1.0, 0.36, 0.51 respectively. Replacing most or all of the Na+ in the AAH caused only a small change in the membrane potential rather than the large hyperpolarization towards the K+ equilibrium potential predicted by the Goldman model. Including the K+ ionophore valinomycin in the low Na(+)-AAH solutions caused a large increase in 86Rb+ efflux but did not result in additional hyperpolarization. This suggested that the insensitivity of the membrane potential to reduced extracellular Na+ was not due to voltage or pH inactivation of lens K+ channels.

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PubMed1992

The control of lens growth: relationship to secondary cataract.

A lens growth factor was identified that is present in the anterior chamber of the embryonic chicken eye. The mitogen is similar to an embryo-specific activity found in embryo serum. Several purified growth factors, applied singly or in combination, did not stimulate cell division in embryonic lens cells. The serum mitogen is a protein which does not bind to heparin-Sepharose. The possibility is presented that the lens epithelium contains two distinct cell types, the proliferating cells of the germinative zone and the mitotically quiescent central epithelial cells. It is proposed that only cells in the germinative zone are capable of responding to normal lens growth factors. It is likely, therefore, that these cells present the greatest risk for secondary cataract formation.

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PubMed1992

Use of a double-label method to detect rapid changes in the rate of cell proliferation.

We developed a double-label method to directly measure the rate at which cells enter S-phase of the cell cycle. All cells in S-phase were first labeled with a short pulse of [3H]-thymidine. This was followed by a longer incubation in bromodeoxyuridine (BrdU), a thymidine analogue. Nuclei labeled with [3H]-thymidine were detected by autoradiography and those labeled with BrdU by immunocytochemistry. Cells labeled only with BrdU must have entered S-phase at some time after the end of the [3H]-thymidine pulse. Thus, the rate of entry of cells into S-phase could be determined. This method was shown to be more accurate and more sensitive than determining changes in the rate at which cells entered S-phase with a continuous labeling protocol. It was possible to detect changes in proliferative activity that occurred in less than 1 hr. We used this double-label technique to study changes in the cell cycle during the terminal differentiation of chicken embryo lens fiber cells. These studies revealed differences in the effects of several treatments known to stimulate fiber cell differentiation. They also demonstrated the presence in the embryonic eye of factors that stimulate and prevent lens cell proliferation and differentiation.

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PubMed1992

Local synthesis and developmental regulation of avian vitreal insulin-like growth factor-binding proteins: a model for independent regulation in extravascular and vascular compartments.

The expression and regulation of insulin-like growth factor-binding proteins (IGFBPs) in developing avian vitreous humor and serum were compared. Vitreal IGF-I-binding activity was highest on embryonic day 6 [E-6; bound/free ratio (B/F), 0.22 +/- 0.019/50 microliters), decreased 10-fold between E-6 and E-19, and then remained stable through the remainder of embryonic development. In contrast, serum IGF-I binding increased 2-fold over this period, from a B/F of 0.380 +/- 0.056 (E-6) to a B/F of 0.89 +/- 0.18 (E-19). After hatching, serum IGF-I-binding activity continued to increase through posthatching week 12, while vitreal IGF-I binding increased only slightly and then remained constant. Although IGF-II binding in the vitreous humor and serum is 2- to 3-fold higher than that of IGF-I, the same pattern of developmental regulation was observed as with IGF-I. Western ligand blots revealed a vitreal 24-kilodalton (kDa) IGFBP that was absent from both embryonic and adult sera. Likewise, posthatching serum was found to contain a 70-kDa IGFBP absent in vitreous humor. Deglycosylation of vitreal and serum IGFBPs followed by Western ligand blotting revealed unique glycosylation patterns for vitreal and serum IGFBPs. One of the IGFBPs that is differentially glycosylated in vitreous and serum is a 33-kDa IGFBP that is precipitated with human IGFBP-2 antiserum. Northern blot analysis revealed the presence of IGFBP-2 mRNA in several embryonic ocular tissues as well as liver. The observations that vitreal and serum IGFBP levels are independently regulated during development and that IGFBPs from these two compartments have different molecular weights and glycosylation patterns suggest that the vitreal IGFBPs are not derived from serum. The presence of IGFBP-2 mRNA in ocular tissue surrounding the vitreal chamber supports the view that certain vitreal IGFBPs may be synthesized locally.

Aging↗

PubMed1991

Messenger RNA stabilization in chicken lens development: a reexamination.

Previous studies, using actinomycin D, suggested that the rate of mRNA degradation in chicken embryo lens epithelial cells decreased sharply between Days 11 and 13 of development (Yoshida and Katoh, Exp. Eye Res. 11, 1971; Exp. Cell Res. 71, 1972). We repeated these studies and directly measured the effects of actinomycin on lens mRNAs. Although actinomycin decreased [3H]uridine incorporation greater than 90%, only modest decreases in methionine incorporation were detected. Treatment for 6 hr had no detectable effect on delta-crystallin mRNA levels or on the relative amounts of proteins synthesized in an in vitro translation system. Unlike the previous studies, we did not find significant changes in the stability of lens epithelial mRNAs during development.

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PubMed1991

Rapid re-amplification of PCR products purified in low melting point agarose gels.

A rapid, simple method is described for performing sequential amplifications of purified products produced by the PCR. After the initial amplification, an aliquot of the reaction is run on a low melting point agarose gel. A Pasteur pipet is used to punch out a gel plug from the amplified band. The DNA in this plug is then used directly as the template for a second round of amplification. Relatively large amounts of agarose can be tolerated without noticeable effects on amplification. Use of a composite gel made from agarose and linear polyacrylamide increases the ease and utility of this technique. These gels are simple to cast, easier to handle and permit several replicate plugs to be obtained from a single band. This method is well suited to experiments which use "nested" primers to increase the sensitivity and specificity of amplification or any method in which PCR amplification follows DNA purification by electrophoresis in LMP agarose gels.

Electrophoresis, Agar Gel↗

PubMed1990

Volume regulation in lens epithelial cells and differentiating lens fiber cells.

Previous studies from our laboratory have led us to conclude that lens cell elongation is caused by an increase in cell volume. This volume increase results from an increase in the potassium content of the cells due to decreased potassium efflux. In contrast, an increase in the volume of most cells triggers a regulatory volume decrease (RVD) that is usually mediated by increased potassium efflux. For this reason, chicken embryo lens epithelial cells were tested to see whether they were capable of typical cell volume regulation. Changes in cell volume during lens fiber differentiation were first estimated by 3H2O water uptake. Cell water increased in proportion to cell length in elongating lens cells. Treatment of epithelial cells cultured in basal medium with dilute or concentrated medium, or with medium containing 50 mM sucrose, resulted in typical volume regulatory responses. Cells lost or gained volume in response to osmotic stress, then returned to their previous volume. In addition, the elongation and increase in cell volume that accompanies lens fiber cell differentiation occurred normally in either hypo- or hypertonic media. This observation showed that the activation of mechanisms to compensate for osmotic stress did not interfere with the increase in volume that accompanies elongation. The ability of elongating cells to volume regulate was also tested. Lens epithelial cells were stimulated to elongate by exposure to embryonic vitreous humor, then challenged with hypotonic medium. These elongating cells regulated their volume as effectively as unstimulated cells. Therefore, cells that were increasing their volume due to reduced potassium efflux could adjust their volume in response to osmotic stress, presumably by increasing potassium efflux. This suggests that the changes in potassium efflux that occur during differentiation and RVD are regulated by distinct mechanisms.

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PubMed1990

Acetylcholinesterase activity as a marker for the development of the ciliary epithelium in the chicken embryo.

Specific histochemical staining for acetylcholinesterase was first visible in the presumptive ciliary epithelium on day 4 of embryonic development. The enzyme did not appear in the neural retina until day 6. The presence or absence of this enzyme activity enabled us to differentiate the presumptive ciliary epithelium from the adjacent presumptive neural retina early in development. In the rat embryo, acetylcholinesterase was not found in the ciliary epithelium at any stage, although its appearance in the neural retina followed a temporal pattern similar to that seen in chicken embryos. Weak AChE staining was seen in the ciliary epithelium of the rat by 15 days after birth. The function of acetylcholinesterase in the developing ciliary epithelium is unknown, but from the data currently available it is unlikely to be involved in the degradation of acetylcholine. Our observations suggest that some previous studies of retinal development may have misinterpreted events occurring in the embryonic ciliary epithelium as characteristics of the peripheral retina.

Acetylcholinesterase↗

PubMed1990

Intracellular pH measurement using single excitation-dual emission fluorescence ratios.

In the present paper, laser spectroscopy was used to evaluate the utility of a new fluorochrome, carboxyseminaphthorhodafluor-1 (Snarf-1), for single excitation-dual emission ratio measurement of intracellular pH (pHi). The emission spectrum of Snarf-1 showed clear pH-dependent shifts, and emission ratios calculated from the 640 and 587 nm maxima were a sensitive indicator of pH. When irradiated in Cunningham chambers, solutions of Snarf-1 were rapidly bleached, and at pH 7.3 or higher, this bleaching led to a decrease in the 640/587 nm emission ratio. These ratio changes were also observed in intracellular measurements on lens embryonic epithelial cells under conditions in which the entrapped dye was rapidly bleached. As the laser dosage was reduced (by increasing the step size between sample points), bleaching could be reduced to very low levels, and under these conditions, the ratio remained constant. Snarf-1 loaded into lens epithelial explants was calibrated intracellularly using nigericin. Intracellular calibration curves were shifted to more alkaline values than in vitro curves. Intracellular calibration allowed estimates of pHi that were in reasonable agreement with previously published values for lens tissue. Potential artifacts arising from differential photobleaching and intracellular-in vitro calibration are discussed.

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PubMed1990

Localization of insulin-like growth factor-1 binding sites in the embryonic chicken eye.

Insulin-like growth factor-1 (IGF-1) binding sites were localized in the embryonic chicken lens, retina, and retinal pigmented epithelium (RPE) with the use of autoradiography. Each of the ocular tissues exhibited specific binding of radiolabeled ligand. Labeling occurred over the entire surface of the lens from 6-day-old embryos, including the epithelium, basal ends of the fiber cells, and the annular pad. There was relatively little labeling over the lens nucleus. A similar pattern was seen in lenses from 19-day-old embryos. In isolated retinas from embryos of this age, the inner and outer plexiform layers were most heavily labeled. In the 19-day-old RPE, only the apical surface of the cells was heavily labeled. Electron microscopic studies revealed an apical layer of membranous material that may represent outer photoreceptor segments that remained attached to the RPE during dissection. It was uncertain, therefore, whether the IGF-1 was binding to sites on the RPE or to these membrane fragments.

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PubMed1989

Cytochalasin prevents cell elongation and increases potassium efflux from embryonic lens epithelial cells: implications for the mechanism of lens fiber cell elongation.

Treatment of cultured embryonic lens epithelial cells with vitreous humor causes them to differentiate into lens fibers. Fiber differentiation is characterized by extensive cell elongation. We have previously suggested that potassium accumulation provides the driving force for the early, rapid phase of this process. Cytochalasin D prevented cell elongation, reduced potassium accumulation and increased potassium efflux from lens epithelial cells treated with vitreous humor. Potassium accumulation was also inhibited and efflux increased by the drug when lens epithelia were cultured in basal medium, in which they do not differentiate. Because vitreous humor appears to cause cell elongation by reducing potassium efflux, cytochalasin may prevent elongation by increasing potassium efflux. The results of this study suggest that the molecules that mediate potassium efflux are linked to the sub-membrane cytoskeleton.

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PubMed1988

Decreased membrane permeability to potassium is responsible for the cell volume increase that drives lens fiber cell elongation.

Differentiation of lens epithelial cells into fiber cells involves an increase in cell volume which previously was proposed to be the direct cause of the extensive cell elongation which accompanies fiber formation. In this study we have continued to investigate the mechanism underlying cell elongation by defining the minimum nutrient and ion requirements of elongating cells, measuring potassium and sodium fluxes in stimulated and unstimulated lens epithelia, and determining the effects of several pharmacological agents on elongation and ion transport. We have shown that elongation will occur in a basic salt/glucose solution with Insulin-like growth factor I/somatomedin-C stimulation. Neither sodium nor any metabolite appears to be the osmotically active species which drives the increase in cell volume. However, potassium efflux rate coefficient was 47% lower in differentiating cells than in unstimulated cells, whereas potassium uptake rates and ouabain effects were similar. Cells did not elongate in potassium-free medium nor in the presence of several drugs which prevent the accumulation of intracellular potassium or hinder osmotic water flux. Unstimulated cells elongated, however, with the application of quinidine, a drug known to block potassium channels. We propose that stimulation of lens epithelial cells with an insulin-like growth factor signals the closure of a certain population of potassium channels. As a result, potassium efflux from the differentiating cells slows while active potassium uptake continues at a constant rate. Potassium then accumulates within the cell causing water influx, an increase in cell volume, and cell elongation.

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PubMed1987

Lentropin, a protein that controls lens fiber formation, is related functionally and immunologically to the insulin-like growth factors.

Lentropin, a factor present in the vitreous humor of the eye, stimulates lens fiber differentiation from chicken embryo lens epithelial cells in vitro. Lentropin has been partially purified but has not been isolated in sufficient quantity or purity for direct comparison with other growth and differentiation factors. Previous studies have shown that insulin and fetal bovine serum share with lentropin the ability to stimulate lens fiber formation from cultured epithelial cells. In the present study, a number of hormones and growth factors were assayed for lentropin activity. Of those tested, the only substances that had this activity were the insulin-like growth factors (IGFs) somatomedin C (Sm-C/IGF-I) and multiplication-stimulating activity (MSA/IGF-II). Sm-C/IGF-I was approximately 30 times more potent than insulin or MSA/IGF-II in promoting fiber cell formation. A monoclonal antibody to human Sm-C/IGF-I inhibited purified Sm-C/IGF-I, fetal bovine serum, and chicken vitreous humor from stimulating fiber cell differentiation in vitro. This antibody has been shown not to crossreact with insulin and did not block insulin-stimulated lens fiber formation. These findings indicate that lentropin is related to the IGFs and that these factors may play important roles in controlling cell differentiation, in addition to their better-known ability to stimulate cell division.

Animals↗

PubMed1986

Localized vascular regression during limb morphogenesis in the chicken embryo. I. Spatial and temporal changes in the vascular pattern.

Morphogenesis of the vertebrate limb bud depends upon reciprocal interactions between epithelial and mesenchymal tissues. A characteristic limb vascular pattern is essential for normal limb outgrowth. The vascular pattern in the distal portion of the wing bud was examined by ink injection and compared to the sites of cartilage differentiation, as indicated by [35S]-incorporation. During development, avascular areas arose in three distinct locations within the vascularized mesoderm. These areas corresponded to the distal skeletal elements, referred to as digits 2, 3, and 4. Incorporation of radioactive sulfate was high in the avascular areas and low in the adjacent vascular tissue. Examination of autoradiographs of ink-injected limbs suggested that the appearance of avascular regions preceded the accumulation of sulfated cartilage matrix. These results indicate that remodeling of the limb vasculature is related to the formation of the skeletal pattern.

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