Nailfold capillary morphology in exfoliation syndrome.

PurposeThe purpose of the study was to investigate nailfold microvascular morphology in exfoliation syndrome with or without glaucoma (XFS/XFG) compared with primary open-angle glaucoma (POAG) and control subjects using nailfold capillary videomicroscopy.Patients and methodsWe used a JH-1004 capillaroscope to perform nailfold capillary videomicroscopy on the fourth and fifth digit of the non-dominant hand. We enrolled 56 XFS/XFG patients, 87 POAG patients, and 75 control subjects. Masked observers graded the videos for hemorrhages, avascular zones ≥200 microns (µm), and degree of microvascular tortuosity on a four-point subjective scale. Multivariable odds ratios, 95% confidence intervals and P-for trends for assessing the relation between morphological changes and POAG or XFS/XFG were obtained from logistic regression analyses. We also assessed this relation with XFS/XFG compared with POAG in multivariable models.ResultsAfter adjusting for multiple covariates, nailfold hemorrhages, avascular zones ≥200 µm, and higher degree of vascular tortuosity were more common in XFS/XFG vs controls (P-for trend ≤0.0001) and in POAG vs controls (P-for trend ≤0.01). For each 100 capillaries, the number of hemorrhages was similar (P-for trend=0.91) between XFS/XFG and POAG patients; however, there were more avascular zones per 100 capillaries with borderline significance (P-for trend=0.04) in the XFS/XFG group. XFS/XFG patients had more tortuosity than POAG patients; specifically, having a tortuosity score ≥1.5 was associated with a 4.4-fold increased odds of XFS/XFG (95% confidence interval: 1.5-13.3) relative to a tortuosity score <1.0 (P-for trend=0.005).ConclusionA high degree of nailfold capillary tortuosity is a distinct non-ocular feature associated with XFS/XFG compared with either POAG or controls.

Ocular hypotensive and aqueous outflow-enhancing effects of AL-3037A (sodium ferri ethylenediaminetetraacetate).

AL-3037A (Sodium ferri ethylenediaminetetraacetate), a novel compound shown to stimulate the degradation of glycosaminoglycans, was evaluated for its effects on aqueous humor outflow and intraocular pressure (IOP) in four experimental models. Its effect on outflow facility was assessed in bovine and human ocular perfusion organ cultures. Its IOP effect was tested in normotensive and dexamethasone-induced ocular hypertensive rabbits. In bovine eyes, perfusion with AL-3037A (0.1% w/v, 2.3 m M) significantly increased the outflow facility well above the normal 'wash-out' effect. At 30 min after perfusion, the outflow facility of drug-treated eyes increased by 26.0+/-2.8% (mean +/- S.E.(M.), n = 8), significantly higher than the 12.1 +/- 2.8% increase in vehicle-treated eyes. This difference sustained throughout the study period (2 hr). The compound also enhanced aqueous outflow in perfused human anterior segments. In non-glaucomatous eyes, it produced a small decrease in IOP (15.4 +/- 4.6%, n = 17), but in tissues derived from glaucoma patients, bolus administration of 3 mg (7 micromol) of AL-3037A lowered the IOP by 52-68% (n = 2) lasting for at least 3 hr. This outflow-enhancing effect of AL-3037A in ex vivo studies was confirmed by in vivo results. In normotensive rabbits, oral (50 mg kg(-1)), intravenous (10 mg kg(-1)), or topical (2 mg; 50 microl of 4% w/v solution) administration of AL-3037A produced maximum reduction of IOP, when compared to vehicle-treated animals, by 34.7+/-3.5% (n = 10), 22.0 +/- 4.6% (n = 10), and 21.6 +/-4.5% (n = 10), respectively. In dexamethasone induced ocular hypertensive rabbits, topical application of the compound (0.5 mg; 25 microl of 2% w/v solution) reduced IOP significantly by 19.2+/- 0.4% (n = 7) at 3 hr after dosing. Importantly, the IOP lowering effect of AL-3037A did not diminish even after repeated treatments in consecutive days. Thus, in the four study models across three animal species, AL-3037A was demonstrated to be an efficacious ocular hypotensive compound whose effect is most likely mediated by augmentation of the aqueous outflow. Its proposed action on the metabolism of glycosaminoglycans may provide a new and unique mechanism for the treatment of glaucoma.

The presence of transcription factors in chicken albumin, yolk and blastoderm.

Embryonic development is determined by preset intrinsic programs and extrinsic signals. To explore the possibility that transcription factors are present at the onset of development, preparations of yolk, albumin, and blastoderm from unfertilized and fertilized white Leghorn chicken eggs were screened by a panel of 16 transcription factor antibodies with Western blot techniques. Yolk was positive for 13 transcription factors, whereas blastoderm was positive for 10, and albumin was positive for 5. In yolk, several transcription factors, GATA-2, E2F-1, MyoD, and TFIID, were developmentally regulated. These results indicate that intracellular yolk and extracellular albumin contain transcription factors which presumably influence early chick embryonic development from prefertilization to the late blastoderm stage. Thus, the utility of preset maternal transcription factors within yolk and albumin complement maternally derived mRNA to determine the early development of the zygote.

The presence of transcription factors in fetal bovine sera.

Three sources of fetal bovine serum (FBS) were fractionated by ammonium sulfate precipitation and by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), transferred to Immobilon-P membranes, immunoblotted with a panel of transcription factor antibodies, and detected by enhanced chemiluminescence. Nine transcription factors were detected--ATF-2, SRE-ZBP, GATA-2, TFIID, Ets-1/Ets-2, E2F-1, Oct-2, p53, and AP-2; four transcription factors were not detected--Myo D, CREB, Sp2, and Wilms' tumor. The results indicated the presence of varying amounts of several transcription factors in three commercial sources and may represent heretofore unrecognized factors influencing cell culture.

CD44H localization in primary open-angle glaucoma.

PURPOSE: Primary open-angle glaucoma (POAG) is associated with a decreased content of hyaluronan in the trabecular meshwork and in the juxtacanalicular connective tissue. In this study, the authors examined selected regions of the anterior segment to localize and determine the content of CD44H, a transmembrane multifunctional glycoprotein and the principal receptor of hyaluronan. METHODS: Sections of ethanol-fixed anterior segments of six POAG and six normal postmortem eyes were analyzed by immunostaining with and without the nonionic detergent Triton X-100, using the CD44H monoclonal antibody, and the avidin/biotin complex. They were visualized by Vector VIP substrate and were quantitated by computer-aided color image analysis. RESULTS: CD44H was expressed in all regions. Statistically significant decreased content of CD44H was observed in the POAG regions compared with normal regions--ciliary muscle (P < 0.001), ciliary stroma (P < 0.001), anterior iris (P < 0.05), iris root (P < 0.05), and trabecular meshwork (P < 0.05)--and in a subgroup of nonlaser POAG juxtacanalicular connective tissue (P < 0.05) and trabecular meshwork (P < 0.01). In sections treated with Triton X-100 a further increase in immunostaining was observed in normal eyes. As evidenced by scattergram plots of the ciliary body stroma region of the change in the optical density of CD44H between pretreatment with Triton X-100 and without Triton X-100 (y axis) versus the optical density of CD44H without Triton X-100 (x axis), individual cases of POAG were separated from normals. CONCLUSIONS: These results indicate that CD44H may represent a marker of POAG and an etiologic factor in the POAG disease process.

Myelomeningocele and Waardenburg syndrome (type 3) in patients with interstitial deletions of 2q35 and the PAX3 gene: possible digenic inheritance of a neural tube defect.

From a spina bifida clinic we have identified two patients with a syndrome of myelomeningocele and Waardenburg syndrome type 3 (WS3). The patients each possess a single, de novo, interstitial deletion of chromosome 2 (2q35-36.2), including the PAX3 gene. Deletion of PAX3 was confirmed by fluorescence in situ hybridization (FISH). Analysis with PAX3 and flanking microsatellites shows that the deleted interval of chromosome 2 is of paternal origin and is at least 2 and 6 cM in the two patients. Interstitial deletions in this region result in the Waardenburg syndrome (WS1), but have not been associated with neural tube defects (NTDs). Although other etiologies have not been formally excluded, these patients raise the possibility of a digenic etiology of their NTDs via a genetic interaction of the deleted PAX3 gene with a second unidentified locus.

Pathological changes in exposed neural tissue of fetal delayed splotch (Spd) mice.

If meningomyelocele is indeed a progressive intrauterine process, then early delivery or possibly intrauterine repair of meningomyelocele becomes an issue. Utilizing the delayed splotch (Spd) mouse, a genetically transmitted neural tube defect model, we looked for evidence of abnormalities of neural tissue exposed to amniotic fluid. Affected embryonic and fetal mice were examined with the light microscope, and also with the transmission and scanning electron microscope. Neuronal development and programmed cell death paralleled normal fetal development. No evidence of inflammation on or within the exposed neural tissue was observed. Because the vascular supply to the alar and basilar plate are different, vascular development was also examined and no difference could be found. In conclusion, we found no evidence of deterioration of the exposed neural tube during the gestational period of a mouse, which suggests that exposure of unneurulated spinal cord to amniotic fluid is not a risk factor to the fetus with a neural tube defect.

Ultrastructural studies of primary congenital glaucoma in rabbits.

BACKGROUND: The cause of congenital glaucoma is unknown. METHODS: To determine whether the site of impaired aqueous outflow is the entrance to the trabecular meshwork (TM), within the TM, the aqueous drainage plexus, or a combination thereof, the process of TM development was examined by scanning and transmission electron microscopy on postnatal day 3 and weeks 1, 2, 3, 4, and 6 in New Zealand rabbits homozygous for the buphthalmic (bu/bu) gene compared with age-matched controls. RESULTS: Openings to the entrance of the TM in congenital glaucoma were observed, and there was no evidence of an endothelial membrane occluding aqueous flow to the TM. The morphology of the congenital glaucoma TM was abnormal in all bu/bu rabbits by 2 weeks and was characterized by a smaller entrance to the TM at the iris base, smaller intertrabecular openings within and between the trabecular lamellae, and at 6 weeks, iris pillars with extensive lateral extensions in the angle recess. Most intertrabecular spaces were open, however, the inner intertrabecular spaces adjacent to the aqueous plexus were compressed. CONCLUSION: These results suggest the development of congenital glaucoma, which involves a mutation in an autosomal recessive gene and leads to loss of function of a gene(s) required for the differentiation of the TM.

Functional opening of the fourth ventricular outlet in C57BL/6J and delayed splotch mouse embryos.

To compare the functional development of the fourth ventricular outlet in the myeloschisis-Chiari malformation complex with that of a normal brain, the chronological development of the outlet in C57BL/6J non-neural-tube defect mouse embryos was examined as the first step. Then we compared the results with those of homozygotic delayed splotch (Spd) mouse embryos which had neural-tube defects (NTDs). Ferrous chloride (Prussian blue) solution was injected into the lateral or mesencephalic ventricle on gestation days 13-16 in the case of control C57BL/6J mouse embryos and on gestation days 14-16 in the case of homozygotic Spd mouse embryos which had open spinal NTDs and hindbrain anomalies comparable to human Chiari malformation. At 30 min after the injection, acid fixative was infused through the heart to set off the Prussian blue reaction, which makes the dye visible by the precipitation of ferric chloride. According to the present method, more than 75% of C57BL/6J mouse (non-NTD control) embryos showed the evidence of function of the fourth ventricular (4V) outlet from gestation day 15. It was difficult to apply the same method to Spd mouse embryos with NTDs due to the small size of ventricles. Only 4 injections were successful, of which 3 showed the functioning evidence of the 4V outlet. Though the number of mouse embryos with NTDs studied was small, the results suggest that the chronological progress of functional opening of the fourth ventricle in mouse embryos with NTDs is similar to that of control non-NTD embryos.

Glycosaminoglycan stratification of the juxtacanalicular tissue in normal and primary open-angle glaucoma.

PURPOSE: The juxtacanalicular tissue (JCT) is the probable site of aqueous outflow resistance in normal eyes and of the increased resistance in primary open-angle glaucoma eyes (POAG). The purpose of this histochemical study was to determine the glycosaminoglycan (GAG) composition and stratification in the JCT of POAG and age-matched normal eyes. METHODS: Five eyes from four normal donors and five eyes from four POAG donors (69 to 80 years of age) were analyzed. Using methods that histochemically preserve GAGs, GAG-degrading enzymes, Alcian blue staining, and real color discrimination to exclude pigment, nuclear staining and unstained areas, the type and amount of GAGs were estimated by compute-raided charge-coupled device color video image analysis. To examine GAG stratification, the JCT was segmented into three regions-anterior, middle, and posterior-to examine regional differences in GAG composition; each region was further divided into four 2-microns layers, from layer 1, adjacent to and including the endothelium of Schlemm's canal, to layer 4, to the first trabecular lamellae. RESULTS: The normal GAG JCT profile was as follows: hyaluronic acid (HA), 7.78 +/- 1.23 femtograms (fg)/micron2; chondroitin sulfates (CS), 8.18 +/- 0.82 fg/micron2; dermatan sulfate, 0.29 +/- 0.18 fg/micron2; the total, 18.73 +/- 0.68 fg/micron2. In contrast, the POAG GAG JCT profile was as follows: HA 0.57 +/- 0.31 fg/micron2 (P < 0.00001), a 93% decrease; CS 13.49 +/- 0.74 fg/micron2 (P < 0.0001), a 83% increase; dermatan sulfate, 0.90 +/- 0.53 fg/micron2; and the total, 17.31 +/- 0.95 fg/micron2, an 8.2% decrease. The HA was depleted in all layers of all regions of POAG JCT. CONCLUSIONS: Results indicate that the normal JCT is stratified, with HA as the predominant GAG in layers 1 and 2. The POAG JCT is depleted of HA and has an accumulation of CS, which may increase outflow resistance and, consequently, increase intraocular pressure in patients with POAG.

Concanavalin-A-induced open neural tube defects in chick embryos.

Open neural tube defects developed in 12 of 122 alive chick embryos treated with exogenous lectin (concanavalin-A) at stages between 10 or 14 as defined by Hamburger and Hamilton. Embryos treated at stage 10, the time of anterior neuropore closure, developed exencephaly or extensive neural openings from the level of rhombencephalon to the thoracic spinal cord, while embryos treated at stages between 11 and 14, at posterior neuropore closure, developed only small myeloschisis in the thoracolumbar region. The failure of neural tube closure at a critical time is a major cause of neural tube defects.

Glycosaminoglycans of the human trabecular meshwork in primary open-angle glaucoma.

PURPOSE: Glycosaminoglycans (GAGs) contribute to the filtration barrier of aqueous outflow through the trabecular meshwork (TM). The purpose of this biochemical study was to identify the type and amount of GAGs in normal and in primary open-angle glaucoma (POAG) TM and adjacent anterior segment structures. METHODS: The GAGs of 21 masked individual normal and POAG human TMs, as well as iris, ciliary body, and anterior sclera, were isolated biochemically, identified by selective GAG-degrading enzymes, and quantitated by computer-enhanced densitometry. RESULTS: In 10 normal TMs (8 donors, 65 to 83 years of age), the GAG profile was: hyaluronic acid (0.77 +/- 0.26 ng/microgram dry-defatted weight +/- SEM); chondroitin 4(6-) sulfates and dermatan sulfate, collectively referred to as chondroitin sulfates (1.90 +/- 0.13 ng); keratan sulfates (0.33 +/- 0.06 ng); heparitin sulfates (2.02 +/- 0.52 ng); GAG enzyme-resistant material (0.02 +/- 0.01 ng); and total GAGs (5.05 +/- 0.70 ng). In 10 POAG TMs (6 donors, 67 to 88 years of age), the GAG profile was: hyaluronic acid (0.18 +/- 0.11 ng; P < 0.02, a 77% decrease; 6 of 10 TMs contained no detectable hyaluronic acid); chondroitin sulfates (2.39 +/- 0.31 ng); keratan sulfates (0.21 +/- 0.06 ng); heparitin sulfates (1.36 +/- 0.43 ng); GAG enzyme-resistant material (0.08 +/- 0.01 ng; P < 0.02); and total GAGs (4.09 +/- 0.33 ng; statistically insignificant). In the POAG iris, hyaluronic acid content was less (82% decrease, P < 0.02), and the chondroitin sulfates content was higher (72% increase, P < 0.02). Similarly, the POAG ciliary body and anterior sclera contained less hyaluronic acid and more chondroitin sulfates. The GAG profile of a "glaucoma suspect" donor specimen was similar to that of the POAG donor specimen. CONCLUSIONS: The data provide the first quantitative biochemical profiles of GAGs of individual normal and POAG TM, and we suggest that a depletion of hyaluronic acid and the accumulation of chondroitin sulfates may increase aqueous outflow resistance in the POAG TM:

Surface-tension properties of hyaluronic Acid.

PURPOSE: The maintenance of flow channels in the trabecular meshwork is dependent, in part, on the patency of the trabecular spaces. Because the amount of hyaluronic acid decreases in the trabecular meshwork of patients with primary open-angle glaucoma, a change in surface tension may be one of the effects of hyaluronic acid on aqueous outflow. METHODS: The surface-active properties of hyaluronic acid (concentration of 0.156-2.5 mg/ml; molecular weights of 100,000, 500,000, and 4,000,000) in deionized water, Ringer's lactate, Ringer's lactate plus 0.06 mg/ml bovine serum albumin, and mock aqueous solution were tested using the drop volume method. RESULTS: At a hyaluronic acid concentration of 0.312 mg/ml, surface tension decreased; at higher concentrations, a further decrease in surface tension was observed. In the presence of Ringer's lactate, the 100,000-MW hyaluronic acid was more active than the 4,000,000-MW hyaluronic acid. In the presence of Ringer's lactate plus bovine serum albumin or mock aqueous solution, the influence of surface tension of the 100,000-MW hyaluronic acid was moderated: with lower hyaluronic acid concentrations, the decline in surface tension was more than with Ringer's lactate, but with higher hyaluronic acid concentrations, the decline in surface tension was less than with Ringer's lactate. At high concentration, hyaluronic acid behaves like a non-Newtonian fluid, becomes more viscous, and may act to "seal" the trabecular space. CONCLUSIONS: The results of this study indicate that hyaluronic acid possesses surface-active properties, which is just one of several properties of hyaluronic acid that may influence aqueous outflow resistance.

Microscale analysis of the glycosaminoglycans of human trabecular meshwork: a study in perfusion cultured eyes.

SUMMARY: Glycosaminoglycans (GAGs) and proteoglycans of the trabecular meshwork may be involved in the pathogenesis of primary open angle glaucoma and of corticosteroid-induced glaucoma. Although numerous studies of GAGs have been performed on trabecular cells in monolayer culture and in animals, few studies have examined intact human meshwork. Using a new analytic method for quantitative determination of GAGs from individual meshworks, we studied the GAGs of human trabecular meshwork in normal eyes and intact meshwork from perfusion-cultured eyes. Eyes in the culture system were treated with dexamethasone for 21 days in an attempt to create a model of steroid glaucoma. The effect of ascorbate, also known to influence GAG levels, was examined in a preliminary study. Cultured trabecular meshwork had a significant decrease in total GAG levels when compared with meshwork from normal, noncultured eyes (1.16 +/- 0.54 ng/mug vs. 3.51 +/- 0.57 ng/mug; p < 0.01). Ascorbate tended to cause a decrease (28%) in the total level of GAGs, although the difference was not significant. No significant difference in mean GAG levels was found between the dexamethasone-treated and fellow control eyes. Dexamethasone was associated with a large biologic variability in total GAG level (coefficient of variation 89%). In an attempt to identify individual steroid responders mixed in with a group of nonresponders, analysis of individual eyes revealed three of nine eyes to have total GAGs 250% than fellow control eyes, although this variability could also be due to other causes. No consistent change in group mean intraocular pressures was found in the dexamethasone-treated eyes. Intraocular pressures were positively correlated with total GAG levels (r = 0.59; p < 0.01).

Synaptic vesicle and synaptic membrane glycoproteins during pre- and postnatal development of mouse cerebral cortex, cerebellum and spinal cord.

Glycoproteins of synaptic vesicles and synaptic membranes play an important role during the process of synaptogenesis. In order to study the temporal expression of specific carbohydrates and the expression of selected neural proteins, we used peroxidase-conjugated lectin overlays on Western blots and immunoblots of synaptic vesicles and synaptic membranes isolated from pre- and postnatal mouse cerebral cortex, cerebellum and spinal cord. Our lectin overlays on Western blots showed that (1) the synaptic vesicle glycoproteins, gp80-100, gp47 and gp44, and (2) the synaptic membrane glycoproteins, gp180, gp72, gp70 and gp34, show temporal regulation of expression of carbohydrate moieties. Quite significantly, gp47 showed a decrease in the vesicles coinciding with an increase in membranes suggesting a shift in localization. Anti neural cell adhesion molecule (N-CAM) antibody cross-reacted with gp180. The developmental expression of synaptotagmin 1, a well characterized glycoprotein of synaptic vesicle, was determined by immunoblots analysis. Anti synaptosomal-associated protein 25 (SNAP-25) antibody immunoblots were performed in order to compare our results with a developmentally regulated synaptosomal protein demonstrating expression coincident with synaptogenesis. Our immunoblot studies showed that (1) N-CAM (gp180) immunoreactivity decrease with development; (2) the expression of synaptotagmin 1 and SNAP-25 increases as development progresses, and (3) synaptotagmin 1 and SNAP-25 show a shift in subcellular localization (from synaptic vesicle to synaptic membrane) during development, thereby indicating that these proteins are first identified in a vesicular fraction. Thus, our data suggest that synaptic vesicle and synaptic membrane glycoproteins show temporal regulation of specific carbohydrates as well as protein expression during development, which may be a key factor to our understanding of the process of synaptogenesis.

The use of ethanol for preserving proteins and glycoproteins of the trabecular meshwork.

We evaluated methods for storage of the trabecular meshwork and irisciliary body for protein and glycoprotein analysis. The trabecular meshwork and irisciliary body of rabbit eyes were microdissected and stored in Laemmli sample buffer, Carnoy's fluid (75% ethanol-25% glacial acetic acid), or 100% ethanol at ambient temperature, 4 degrees C, -20 degrees C, or -80 degrees C for 24 h or 30 days. Fresh and stored tissues were processed for one-dimensional polyacrylamide gel electrophoresis (PAGE) and Western blot using Con A lectin. The protein patterns of stored and fresh tissues as determined by silver-stained polyacrylamide gels were similar. However, ethanol-stored tissues revealed other proteins (MW of 15-30 kD and 150 kD), and the staining intensity and band resolution of lower MW (15-40 kD) were enhanced. The glycosylation patterns of stored and fresh tissues as determined by Con A (recognizes certain N-linked glycoproteins containing mannose and glucose) were also similar, but the ethanol-stored tissues stained more intensely, especially the high (>200 kD) and low (<35 kD) MW ranges. These PAGE results indicate that ethanol storage is useful for preserving and resolving the protein/glycoprotein profiles of the trabecular meshwork.

Ultrastructural alterations in the aqueous outflow pathway of adult buphthalmic rabbits.

The aqueous outflow pathway of adult rabbit eyes with congenital glaucoma (buphthalmos) was examined by light microscopy and by scanning and transmission electron microscopy. The morphology of the buphthalmic rabbit aqueous outflow pathway was markedly abnormal when examined at 6 months, 1 yr, and 2 yr displaying apparent loss and/or compression of the iris pillars, dilation of the intertrabecular spaces, loss of endothelial cell-to-cell association and disorganization of trabecular lamellae, and posterior displacement of the aqueous plexus. In addition, the trabecular meshwork lamellae were observed only adjacent to the sclera and the inner portion of the trabecular meshwork was limited to swirls of collagen with scattered cells. These morphological findings suggest that the disease process in the rabbit principally involves an alteration in the differentiation and maintenance of the structural integrity of the trabecular meshwork. The loss of structural support of the buphthalmic trabecular meshwork may be a factor in the wide variation in intraocular pressure and may allow for compression of the trabecular meshwork against the aqueous plexus.

The cause of Chiari II malformation: a unified theory.

The cause of the Chiari II hindbrain deformity in children born with a myelomeningocele can be explained by the lack of distention of the embryonic ventricular system. Defective occlusion and an open neural tube precludes the accumulation of fluid and pressure within the cranial vesicles. This distention is critical to normal brain development. The small posterior fossa, cerebral disorganization, and lückenschädel are the result.

Fluorescence-labeled lectins, glycoconjugates, and the development of the mouse AOP.

The development of the aqueous outflow pathway (AOP) in early postnatal mouse eyes was examined for the presence of a variety of lectin receptors using fluorescein isothiocyanate (FITC) conjugated lectins, 1 micron araldite plastic sections, and computer-aided fluorescence photography. The trabecular meshwork anlage (days 1-4) was characterized by the presence of loosely arranged cells and an extracellular matrix that exhibited intense areas of Con A- and RCA-lectin staining, and absence of WGA- and LPA-lectin staining. By day 6, trabecular meshwork LPA- and WGA-positive materials were observed as focal areas of staining. By day 10, LPA- and WGA-positive materials were present as diffuse areas of staining, as the AOP differentiated into an organized and functional biological filter. The age-dependent pattern of LPA- and WGA-positive materials indicated that there were time-dependent points in the synthesis of glycoconjugates in the developing AOP. The results suggest: The composition and/or conformation of the glycoconjugates on cells and extracellular matrix changed as the AOP differentiated into a functional tissue. The use of FITC lectins as biological markers for studies of the AOP provided information on the potential role of glycoconjugates in the development of the normal AOP. Modification in the type, amount, and distribution of glycoconjugates may provide a basis for understanding the cellular mechanisms of abnormal development of the AOP, eg, congenital glaucoma.

Vitamin A-induced suppression/enhancement of protein glycosylation and neurulation.

Glycoconjugates play major roles in many cellular functions, e.g. cell migration and cell-to-cell adherence, which are involved in neurulation. The maternal administration of vitamin A on gestation day 8.5 and 9.0 resulted in a high percentage of primary and secondary neurulation defects in gestation day 12 mouse embryos. The neuroepithelium of normal and abnormal embryos was analyzed by one- and two-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis and one-dimensional Western blots using concanavalin A (Con A) and peroxidase-conjugated wheat germ agglutinin (WGA) lectins. In vitamin A abnormal embryos, WGA binding was decreased to glycoproteins with apparent molecular weights of 15,000 and 30,000 daltons on Western blots, whereas in vitamin A normal embryos, WGA binding was increased to these glycoproteins on Western blots. Computer-aided fluorescence microscopy using fluorescein isothiocyanate (FITC)-conjugated lectins on 1-micron araldite plastic sections indicated a decrease in FITC-WGA binding to the free surface of nonneurulated neuroepithelium. These results suggest: (1) vitamin A administration may have induced a suppression of WGA-binding carbohydrate residues on 15,000- and 30,000-dalton glycoproteins in abnormal embryos, and (2) modification in the type, amount, and distribution of glycoconjugates may provide a basis for the cellular mechanisms of abnormal development of the neural tube.