Human gene for the RNA polymerase II seventh subunit (hsRPB7): structure, expression and chromosomal localization.

The human gene for the seventh largest subunit of RNA polymerase II complex, hsRPB7 was cloned, sequenced and mapped. This complex is an integral part of the transcription-coupled DNA repair mechanism and has been shown to be involved in several human genetic diseases and implicated in many others. The hsRPB7 gene consists of 8 exons and spans approximately 5.1 kb. Southern blots of genomic and cloned DNA suggest that hsRPB7 is coded for by a single gene. Using human radiation hybrids and YACs, the gene was localized to 11q13.1, within 70 kb of marker D11S1765. The sequence of the 5' flanking region does not contain a TATA element, but does contain several Sp1 binding sites, an AP-1 site and a novel inverted polymorphic GATA tandem repeat. This novel GATA repeat can be used for linkage analysis. The hsRPB7 gene seems to be highly conserved among eukaryotic species, showing general sequence conservation to yeast and Drosophila. Northern blot analysis reveals a high degree of tissue-specific expression. For example, adult retina, brain and kidney exhibit a relatively high level of expression. A moderate level of expression is observed in heart, lung, testis, cornea, retinal pigmented epithelium/choroid and placenta with a lower level of expression in the uterus, small intestine and skeletal muscle. A very low level of expression was observed in stomach and liver. Comparison between four fetal and adult tissues also demonstrate a surprising level of developmental specificity. Expression in fetal retina is considerably lower than fetal brain but similar to adult retina.

Isolation and expression of homeobox genes from the embryonic chicken eye.

PURPOSE: To identify homeobox-containing genes that may play a role in the differentiation of ocular tissues. METHODS: Total RNA was isolated from microdissected chicken embryo eye tissues at 3.5 days of development (embryonic day 3.5; E3.5). An "anchor-oligo-dT primer" was used for the synthesis of cDNA. Degenerate oligonucleotides designed from highly-conserved sequences in the third helix of the homeobox and the "anchor-primer" were used to amplify cDNAs by polymerase chain reaction (PCR). PCR products were cloned and sequenced. The spatial and temporal expression of selected transcripts was mapped by whole-mount in situ hybridization and northern blot analysis. RESULTS: After sequencing eighteen clones we identified a member of the distal-less family (dlx-3) in cDNA from presumptive neural retina and three chicken homologs of the Xenopus "anterior neural fold" (Xanf-1) in cDNA from anterior eye tissue. Dlx transcripts were mapped by in situ hybridization. Expression began at Hamburger and Hamilton stage 14 (E2.5) and was widely distributed in embryonic mesenchyme on E3 and E4. Expression increased in the retina during early development and persisted until after hatching. The one anf clone selected for further study was not detected by in situ or northern blot analysis. CONCLUSIONS: It is feasible to isolate homeobox cDNAs directly from microdissected embryonic tissues. Chicken dlx-3 mRNA has a wider distribution in the embryo than expected, based on the expression of the mouse homolog. Dlx-3 may play a role in establishing or maintaining the differentiation of the retina.

Identification of components of a phosphoinositide signaling pathway in retinal rod outer segments.

Phototransduction in retinal rods involves a G protein-coupled signaling cascade that leads to cGMP hydrolysis and the closure of cGMP-gated cation channels that are open in darkness, producing a membrane hyperpolarization as the light response. For many years there have also been reports of the presence of a phosphoinositide pathway in the rod outer segment, though its functions and the molecular identities of its components are still unclear. Using immunocytochemistry with antibodies against various phosphoinositide-specific phospholipase C (PLC) isozymes (beta1-4, gamma1-2, and delta1-2), we have found PLCbeta4-like immunoreactivity in rod outer segments. Similar experiments with antibodies against the alpha-subunits of the G(q) family of G proteins, which are known to activate PLCbeta4, have also demonstrated G(alpha11)-like immunoreactivity in this location. Immunoblots of total proteins from whole retina or partially purified rod outer segments with anti-PLCbeta4 and anti-G(alpha11) antibodies gave, respectively, a single protein band of the expected molecular mass, suggesting specific labelings. The retinal locations of the two proteins were also supported by in situ hybridization experiments on mouse retina with probes specific for the corresponding mouse genes. These two proteins, or immunologically identical isoforms, therefore likely mediate the phosphoinositide signaling pathway in the rod outer segment. At present, G(alpha11) or a G(alpha11)-like protein represents the only G protein besides transducin (which mediates phototransduction) identified so far in the rod outer segment. Although absent in the outer segment layer, other PLC isoforms as well as G(alpha q) (another G(q) family member), are present elsewhere in the retina.

Developmental changes in serum insulin-like growth factor-I and insulin-like growth factor binding proteins in the turkey embryo.

The ontogeny of insulin-like growth factor-I (IGF-I) and ontogeny as well as the molecular nature of the insulin-like growth factor binding proteins (IGFBP) as they relate to embryogenesis and posthatch growth in the turkey have not been reported. In this study, serum samples were harvested from turkey embryos incubated under shell-less and shelled conditions from Day 12 to 28 of incubation. Samples from 3, 6, and 8 wk posthatch were also evaluated. Significant changes in serum IGF-I in shelled embryos were observed in that IGF-I was low in early incubation, peaked at mid-incubation, and returned to levels equivalent to early incubation at hatching. No mid-embryogenesis (Days 14 to 18) increase in serum IGF-I was noted in the shell-less cultures. Embryo weights diverge at Day 18 of incubation, with shell-less embryos being significantly lighter than their age-matched shelled embryos, suggesting a possible relationship between total circulating IGF-I and body weight gain. Three distinct IGFBP were identified in serum from turkey embryos, exhibiting molecular weights of 27, 29, and 69 kDa. During embryonic development, the 29 kDa IGFBP appeared to be the predominant species, with levels peaking on Day 24 of incubation, and being minimally detectable at hatching. Ontogeny of the 29 kDa IGFBP was similar in the shelled and shell-less embryos. The 69-kDa IGFBP-like protein did not appear in the circulation until late in embryogenesis, Day 22 in shell-less and Day 26 in shelled embryos. The 27-kDa IGFBP appeared in late incubation. Similar IGFBP (28, 30, and 69 kDa) were observed in the growing male turkey. The 69-kDa protein did not vary across ages studied, whereas the 28-kDa IGFBP appeared to increase with age. This is the first report describing serum IGF-I and IGFBP in the developing turkey. Turkey IGFBP appear to be regulated by independent events during incubation and posthatch growth.

Structural organization and chromosomal localization of the human ribosomal protein L9 gene.

The intron-containing gene for the human ribosomal protein L9 has been cloned, sequenced and localized. The gene is approximately 5.5 kb in length and contains 8 exons. Splice sites follow the AG/GT consensus rule. The message for human rpL9 is 712 nt in length and is detected in all tissues examined. In the adult, expression is highest in retina and liver while brain shows highest expression among the fetal tissues tested. The transcription start site contains an oligopyrimidine tract, TTCTTTCTT, similar to those found in other ribosomal protein genes. As in other previously characterized ribosomal protein genes, a TATA box is absent from the 5' flanking region but a number of elements recognized by common transcription factors are present including Sp1 sites, CACCC boxes, inverted CCAAT boxes, and GATA elements. Another possible element of interest in the rpL9 5' flanking region is RFX1 also found in the well characterized rat rpL30 promoter. The gene was mapped by fluorescent in situ hybridization to band 13p of chromosome 4. At least 8 possible pseudogenes are present in the human genome, one of which is on Xp. As assessed by Southern 'Zoo-blot' analysis and direct cDNA sequence comparison, the human ribosomal protein L9 gene, like other ribosomal protein genes, is highly conserved among mammals.

Differential temporal and spatial expression of insulin-like growth factor binding protein-2 in developing chick ocular tissues.

PURPOSE: To determine the developmental expression and localization of mRNA for insulin-like growth factor binding protein-2 (IGFBP-2), a major binding protein of IGF-I and IGF-II, in ocular tissues of the embryonic and early posthatched chick. METHODS: In situ hybridization and northern blot analysis were used to analyze the cellular origin and relative expression of IGFBP-2 mRNA in ocular tissues. RESULTS: Wholemount in situ hybridization reveals that, as early as 3.5 days of embryonic development (E3.5), IGFBP-2 mRNA is already expressed in many areas of the embryo, including surface ectoderm, certain regions of the brain, pharyngeal clefts, somites, and limb buds. In the eye, IGFBP-2 mRNA is expressed only in the presumptive corneal epithelium at this time. By E6, IGFBP-2 mRNA expression is present in both the corneal epithelium and endothelium. By E12, IGFBP-2 mRNA is detected clearly in the corneal stroma as well as in several other ocular structures, such as the sclera, eyelid, and ciliary body. In the neural retina, a low, diffuse expression of IGFBP-2 mRNA is found at E6, which becomes more localized to the nuclear layers by E12. Northern blot analysis confirms that a high level of IGFBP-2 expression is present in the cornea and sclera by E8 to E12. A high level of IGFBP-2 mRNA expression, however, is not observed in the retina until E18. At posthatch day 2 (P2), northern blot analyses of ocular tissues reveal that the cornea contains the highest ocular level of IGFBP-2 mRNA expression, a value equal to that of brain and liver. CONCLUSIONS: The early appearance, along with differential temporal and spatial expression of IGFBP-2 mRNA in developing ocular tissues, suggests a role for IGFBP-2 in the regulation of growth and differentiation of several ocular tissues, including the cornea, sclera, and retina.

Isolation of candidate genes for macular degeneration using an improved solid-phase subtractive cloning technique.

An improved solid-phase subtraction procedure was developed to generate a readily amplifiable library of short cDNA fragments highly enriched in the macula (target) versus the peripheral region (driver) of the monkey neural retina. The generated clones were sequenced and 63 were analyzed by northern blotting using total RNA from the monkey macula and peripheral retina. The results indicate that 32% are highly enriched in macula, 36% are below the limits of detection and 32% are not enriched. No clones were found which were enriched in the peripheral retina. Our technique is therefore successful in identifying novel cDNAs enriched in the macula area of the neural retina that may represent potential candidate genes for hereditary ocular diseases. It should thus be useful in other situations where subtle differences in expression between cell types or tissue areas need to be analyzed.

Cloning and characterization of a chick embryo cDNA and gene for IGF-binding protein-2.

We have isolated and characterized a cDNA for IGF-binding protein-2 (IGFBP-2) and its gene from the chick embryo. Using primers from a conserved region of the mammalian IGFBP-2 sequence, a cDNA clone (1.6 kb) was isolated from an embryonic day-18 chick retina cDNA library. Although the clone was truncated at the 5' end, the complete coding sequence was obtained from 5' rapid amplification of cDNA ends and genomic sequencing. The open reading frame encoded a 311 amino acid precursor protein which contains a putative 36 residue signal peptide. The mature 275 amino acid protein had a predicted M(r) of 33,500 and exhibited 71, 68, 68 and 66% identity to rat, bovine, ovine and human IGFBP-2 cDNA respectively, with conservation of all 18 cysteines. The cDNA contained an RGD peptide but lacked a putative ATP-binding motif. A single transcript of approximately 2.3 kb was present in embryonic day-15 eye, brain, skeletal muscle, heart and intestine, but was virtually absent from embryonic day-15 liver. The chicken IGFBP-2 gene spanned approximately 38 kb, consisted of four exons, and was similarly organized to that of the rat and human. Southern blot analysis of chicken genomic DNA suggested that it is encoded by a single gene. The sequence information from the avian IGFBP-2 should be of value in examining the role of IGFBP-2 in vertebrate development.

Vitreous and aqueous humors contain a latent proteinase activity that abolishes IGF binding to specific IGF binding proteins.

Several distinct insulin-like growth factor binding proteins (IGFBPs) are present in tissues and fluids of the developing and adult eye. However, the mechanism(s) involved in the regulation of ocular IGFBP levels is unknown. We have now identified an endogenous factor in vitreous and aqueous humors that, when activated by sodium dodecyl sulfate (SDS), abolishes the capacity of specific low molecular weight IGFBPs (i.e. 24-30 kDa) to bind IGF as assessed by western ligand blotting. In contrast, IGF binding to the 46 and 32 kDa IGFBPs (IGFBP-3 and IGFBP-2 respectively) is not affected by the SDS-activated inhibitory factor (IF). Maximal activation of the IF occurs at an SDS concentration of approximately 0.015%. Incubations in the presence of the serine-proteinase inhibitor aprotinin result in marked inhibition of IF activity. Preliminary characterization by ultrafiltration suggests that the IF is large (< 100 kDa) and/or that it is present in a complex. The finding of a factor, most likely a serine proteinase, that specifically abolishes IGF binding to low molecular weight IGFBPs suggests a mechanism for regulating the levels of these IGFBPs and thus the functional activities of IGFs in ocular fluids under normal and/or pathological conditions.

Structural organization and expression of the human phosphatidylinositol-specific phospholipase C beta-3 gene.

We have cloned and fully sequenced the phospholipase C beta-3 (PLC beta-3) gene. The gene spans approx. 17 kb and consists of 31 exons and 30 introns. All intron-exon junctions obey the GT/AG rule. The gene is highly expressed in several human tissues including retina, brain and kidney; PLC beta-3 mRNA is detected at a much lower level in liver. Because of its importance in signal transduction, its chromosomal localization and its high expression in CNS and other tissues, the PLC beta-3 gene is a candidate in several human genetic diseases which, with the present genomic sequence, can now be fully examined.

Identification and partial characterization of a proteinase specific for insulin-like growth factor binding protein-3 in aqueous and vitreous humors.

The IGFs (-I and -II) are normally found in serum and other extracellular fluids complexed to specific binding proteins (IGFBPs). While several IGFBPs have been identified in vitreous and aqueous humors, the major serum carrier of IGF, IGFBP-3, is notably absent from these fluids. To determine if this paucity could be due to an IGFBP-3 proteinase (IGFBP-3ase), samples of bovine vitreous or aqueous humor were mixed with serum and incubated at 37 degrees C for 4 h followed by western ligand blotting. In these experiments, a distinct loss of the 46 kDa band representing IGFBP-3 was observed while other bands present at 35, 28 and 25 kDa were unaltered. The IGFBP-3ase activity is temperature sensitive, has a pH optimum of about 8.0 and is inhibited by EDTA. Acid treatment of serum to remove endogenously bound IGF does not affect the specificity or activity of the IGFBP-3 proteinase. Size exclusion chromatography of bovine aqueous indicates an approximate molecular weight of 260 kDa. Incubation of recombinant IGFBP-3 or serum with partially-purified IGFBP-3ase results in the appearance of low molecular weight fragments of approximately 30 kDa. These fragments are undetectable by western ligand blotting but are readily visualized using an IGFBP-3 specific antibody. Comparison of normal and diabetic vitreous humor reveals the presence of an increased amount of IGFBP-3 proteolytic fragments in the diabetic as compared to control. These findings indicate the presence of a IGFBP-3 proteinase in aqueous and vitreous humors that may be important in regulating ocular homeostasis.

Distal versus proximal electrode placement in the prediction of total body water and extracellular water from multifrequency bioelectrical impedance.

In 48 normal weight subjects, 25 females and 23 males, body impedance was measured at multiple frequencies. Two different electrode placements were used, one the commonly used distal electrode placement, in which the source electrodes are on the dorsal sides of the hand and foot and the sensor electrodes are on ankle and wrist, and a second placement, in which the sensor electrodes are placed more proximally, at the knee and elbow. Theoretically a proximal electrode placement could result in more precise estimates of body water compartments. Total body water (TBW) and extracellular water (ECW) were determined using deuterium oxide dilution and bromide dilution, respectively. The aim of the study was to investigate whether proximal electrode placement results in a more precise estimation of TBW and ECW using multifrequency impedance analysis. Correlation coefficients of impedance and the impedance index stature2 /impedance) with TBW and ECW were not or were only slightly higher using proximal impedance values, resulting in slight improvement of the estimation error for TBW (0.13 kg) and ECW (0.04 kg). The differences between measured and predicted values (residuals) of TBW and ECW were not correlated with TBW and ECW, but they were correlated with body fat and body water distribution (ECW/TBW). These correlations did not differ between distal and proximal impedance measurements. It is concluded that proximal impedance measurements do not substantially improve the prediction of body water compartments. © 1995 Wiley-Liss, Inc.

Structural analysis of the human hydroxyindole-O-methyltransferase gene. Presence of two distinct promoters.

Hydroxyindole-O-methyltransferase (HIOMT) catalyzes the last step in the metabolic pathway that synthesizes the hormone melatonin. We have found HIOMT mRNA present in small amounts in human retina and in relatively high abundance in the pineal gland. Two distinct 5' ends were found in human retina using a solid-phase 5'-rapid amplification of cDNA ends technique. The two 5' regions appear to originate from two distinct putative promoters. Although many similarities exist between the two promoters, they contain distinctive elements. Putative promoter A, for example, contains a recently discovered photoreceptor-conserved element (PCE-1, CAATTAAG) at -27 not found in promoter B, while promoter B contains an Ap1 site (ATGAGTCAA) at -166 and an octamer site (ATGCAAT) at -59 not found in promoter A. The HIOMT messages are also alternatively spliced in between exons 6 and 8, generating three distinct messages. One of the alternatively spliced messages contains a line-1 repetitive element that is spliced into the mRNA precisely as exon 6. Importantly, the downstream open reading frame is not altered by any of these splicing combinations. The gene is approximately 35 kilobases long containing either 9 or 10 exons (including the line-1 element) depending on which promoter is active. All of the splice sites follow the GT/AG rule. The dual promoters and opportunities for alternative splicing suggest a variety of mechanisms for control of HIOMT expression and biological activity in different tissues not previously recognized.

Vitreal insulin-like growth factor binding proteins (IGFBPs) are increased in human and animal diabetics.

Although patients with diabetic retinopathy have been reported to have elevated vitreal IGF-I levels, it is not known whether diabetes also affects the levels of vitreal IGF binding proteins (IGFBPs) which control IGF's bioavailability. To address this issue, vitreal IGFBP levels were assayed in human diabetics, rats with streptozotocin-induced diabetes and galactose-fed dogs with diabetic-like retinopathy. Using 125I-IGF-II ligand blots, it was found that human diabetics have a 4-fold increase in vitreal IGFBP levels. Also, western blots on human diabetic vitreous reveal increased levels of IGFBP-2 and proteolytic fragments of IGFBP-3. IGF binding assays on vitreous from streptozotocin-treated rats (three months in duration) also indicate a 5-fold increase in IGF binding activity. IGF ligand blots using vitreous from rats with a shorter duration of diabetes (one month) show a 63% increase in IGFBP binding and a marked decrease in serum IGFBP binding. IGF ligand blots and IGFBP-2 and -4 western blots using vitreous from galactose-fed dogs with diabetic-like retinopathy exhibit a 6-fold increase in vitreal IGFBPs. The observation that vitreal IGFBPs are elevated in diabetic humans and rats without overt retinopathy suggests that these increases are not the result of a preexisting end-stage retinopathy but rather are an early ocular event in the diabetic process. Increases in vitreal IGFBPs thus could participate in the proliferative aspects of diabetic retinopathy by virtue of their putative intrinsic bioactivity or their capacity to alter IGF bioavailability.

Distribution of IGF-I and -II, IGF binding proteins (IGFBPs) and IGFBP mRNA in ocular fluids and tissues: potential sites of synthesis of IGFBPs in aqueous and vitreous.

Levels of insulin-like growth factor-I and II (IGF-I and IGF-II) in bovine aqueous humor are twice those found in the vitreous (aqueal IGF-I = 0.62 nM, vitreal IGF-I = 0.30 nM; aqueal IGF-II = 0.028 nM, vitreal IGF-II = 0.017 nM). IGF-I and II binding assays and IGF-II Western ligand blots indicate that aqueous and vitreous humor have equal overall levels of binding (binding assays, mean +/- S.E.M. bound/free per 50 microliters of fluid: vitreal IGF-II = 7.28 +/- 1.6, IGF-I = 0.3 +/- 0.078; aqueal IGF-II = 7.21 +/- 0.072; IGF-I = 0.3 +/- 0.078). In addition, the ligand blots reveal that aqueous and vitreous have markedly different complements of specific IGF binding proteins (IGFBPs). Aqueal levels of a 34 kDa IGFBP, immunologically identified as IGFBP-2, exceed those in the vitreous by two-fold. In contrast, the vitreous exhibits a two- to three-fold higher level of smaller (28-24 kDa), yet unidentified, IGFBPs. Aqueal and vitreal IGFBP patterns are also different from those found in serum. IGFBP-2 found in the aqueous and vitreous may be synthesized by ciliary body and/or cornea since these structures contain high levels of IGFBP-2 mRNA. Lens epithelial cells may also contribute IGFBP-2 to the aqueous since they also contain IGFBP-2 mRNA, albeit at substantially lower levels than the cornea and ciliary body. The retina has the lowest level of IGFBP-2 mRNA. IGF-II binding assays of cornea, ciliary body, retina and retinal pigment epithelium (RPE) indicate that the cornea has the highest level of binding (mean +/- S.E.M. IGF-II B/F per 50 micrograms protein: cornea = 84.52 +/- 28.8; iris/ciliary body complex = 0.61 +/- 0.078; retina = 0.47 +/- 0.096; RPE = 0.069 +/- 0.019). IGF-II ligand blots confirm these tissue-specific differences in binding and show that each ocular tissue contains IGFBP-2. In addition, ligand blots indicate that each ocular tissue contains a complex and distinctive population of IGFBPs. For example, the cornea and retina (but not the ciliary body, aqueous or vitreous) contain a 46 kDa IGFBP that may be IGFBP-3. The finding that cornea and retina also contain IGFBP-3 mRNA suggests that these structures may synthesize IGFBP-3 for local use within the eye.

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.

Monkey retinal pigment epithelial cells in vitro synthesize, secrete, and degrade insulin-like growth factor binding proteins.

Cultured monkey retinal pigment epithelial (RPE) cells rapidly secrete large amounts of insulin-like growth factor binding proteins (IGF-BPs). IGF-II tracer binding activity in conditioned media is two to three times greater than that of IGF-I. Under reducing SDS-PAGE conditions, 125I-IGF affinity-crosslinked binding protein (BP) is visualized as a broad band between 36 +/- 2.9 and 49 +/- 3.3 kDa. Because the electrophoretic mobility of the crosslinked BP is increased under non-reducing conditions (33-45 kDa), intramolecular sulfhydryl bonding may be present. Frequently, the radiographic band representing affinity-crosslinked binding protein exhibits a complex pattern of non-uniform densities that suggests structural or functional IGF-BP micro-heterogeneity. IGF-BPs synthesized by RPE also exhibit heterogeneity with respect to the absence or presence of oligosaccharide side chains. In particular, the larger, but not the mid-sized or smaller IGF-BPs exhibit side chains linked to the core protein with N-glycosidic linkage. None of the crosslinked IGF-BPs exhibit O-linked side chains. Long-term (12, 24, 48 hr) conditioning studies revealed that IGF-BP fails to accumulate in culture media beyond 12 hr, but that replacement of conditioned media with fresh media allows a second period of binding protein accumulation. Other short-term (12 hr) experiments indicate that, in fresh medium, the levels of IGF-BP increase during the first 6-8 hr and then remain stable. To examine the processes contributing to these steady state levels of IGF-BP, aliquots of 8-hr conditioned medium were removed from the cells and either frozen on dry ice or incubated at 37 degrees C for 16 hr. Importantly, it was found that incubation at 37 degrees C resulted in a near total loss of binding activity. This is the first report of IGF-BP degrading activity in a cell culture system. These findings indicate that 1) primate RPE cells rapidly secrete a complex mixture of N-glycosylated and non-glycosylated IGF-BPs, and 2) the steady state levels of secreted IGF-BP are tightly regulated at least in part through a concomitant IGF-BP inactivating activity. Cultured RPE cells may be of utility in examining the mechanisms of IGF-BP synthesis, secretion, and degradation at the cellular level.

Evidence for an insulin-like growth factor autocrine-paracrine system in the retinal photoreceptor-pigment epithelial cell complex.

The interphotoreceptor matrix (IPM), lying between retinal photoreceptor and pigment epithelial (RPE) cells, contains insulin-like growth factor I (IGF-I) immunoreactivity that co-elutes with authentic human IGF-I in HPLC analyses. Cultured human RPE cells synthesize and release IGF-I, raising the possibility that the RPE serves as a source of IPM IGF-I in vivo. Photoreceptor rod outer segments and cultured monkey RPE cells express specific IGF-I receptors with alpha-subunits of 120 and 138 kDa, respectively. They thus appear to be of the "brain" (in photoreceptors) and "peripheral" (in RPE cells) receptor subtypes. Additionally, the IPM contains high levels of an IGF binding protein (IGF-BP) that specifically binds IGF-I and IGF-II. The IPM-BP is visualized as a single radiographic band by both ligand blot and affinity cross-linking procedures. With enzymes specific for removing N- and O-linked oligosaccharides, the IPM-BP was found to contain O- but not N-linked glycosylated side chains. The distinctive size and glycosylation pattern of the IPM-BP indicate that it is not derived from the vitreous or serum but instead is synthesized locally. The presence of IGF-I and IGF-BP in the IPM, together with the presence of IGF-I receptors on both photoreceptor and RPE cells, suggests the presence of an outer retina autocrine-paracrine system.

Experimental ischemia induces cell mitosis in the adult rat retina.

Experimental occlusion of the central retinal vessels in albino rats followed by reperfusion for 2 or 4 days resulted in a highly active proliferative response. Mitotic figures were produced in retinal vascular cells, presumed Müller cells, retinal pigment epithelial cells, and possibly other cell populations in the retina. A large number of mitoses were observed in 60-100% of 50 experimental eyes with 2, 4 or 6 hr of experimental ischemia and 2 or 4 days of reperfusion and in none of the 37 control eyes. Autoradiography demonstrated 3H-thymidine uptake in the retina and in vascular cells on a trypsin digest preparation of the retina. Retinal ischemia followed by reperfusion leads to mitosis of retinal cells. This suggests a causal relationship in proliferative retinopathies where ischemia and cell proliferation coincide.