Minisatellite diversity supports a recent African origin for modern humans.

In a study of human diversity at a highly variable locus, we have mapped the internal structures of tandem-repetitive alleles from different populations at the minisatellite MS205 (D16S309). The results give an unusually detailed view of the different allelic structures represented on modern human chromosomes, and of the ancestral relationships between them. There was a clear difference in allelic diversity between African and non-African populations. A restricted set of allele families was found in non-African populations, and formed a subset of the much greater diversity seen on African chromosomes. The data strongly support a recent African origin for modern human diversity at this locus.

In vivo biocompatibility of a new cyanine dye for ILM peeling.

PURPOSE: To investigate the biocompatibility of the new cyanine dye: 3,3'-Di-(4-sulfobutyl)-1,1,1',1'-tetramethyl-di-1H-benz[e]indocarbocyanine (DSS) as a vital dye for intraocular application in an in vivo rat model and to evaluate the effects of this dye on retinal structure and function. METHODS: DSS at a concentration of 0.5% was applied via intravitreal injections to adult Brown Norway rats with BSS serving as a control. Retinal toxicity was assessed 7 days later by means of retinal ganglion cell (RGC) counts, light microscopy, optical coherence tomography (OCT), and electroretinography (ERG). RESULTS: No significant decrease in RGC numbers was observed. No structural changes of the central retina were observed either in vivo (OCT) or under light microscopy. ERGs detected a temporary reduction of retinal function 7 days after injection; this was no longer evident 14 days after injection. CONCLUSIONS: DSS showed good biocompatibility in a well-established experimental in vivo setting and may be usable for intraocular surgery as an alternative to other cyanine dyes. In contrast to indocyanine green, it additionally offers fluorescence in the visual spectrum. Further studies with other animal models are needed before translation into clinical application.

alpha B-crystallin in the primate ciliary muscle and trabecular meshwork.

The presence of alpha B-crystallin, a protein with heat-shock protein-like properties, has been demonstrated in ciliary muscle and trabecular meshwork derived from human and monkey eyes using immunohistochemical and polymerase chain reaction methods. Both frozen sections and cultured cells have been analyzed. In the ciliary muscle, alpha B-crystallin staining is localized in the region of the dense bands, in the cytoplasm of the muscle cells and in the Schwann cells of the nerves supplying the muscle. In the trabecular meshwork, two cell types could be distinguished on the basis of alpha B-crystallin occurrence. Whereas the trabecular cells covering the lamellae were virtually devoid of the protein, the subendothelial or cribriform cells contained relatively large amounts in parallel with a higher alpha B-crystallin mRNA level.

Cis-regulation of inter-allelic exchanges in mutation at human minisatellite MS205 in yeast.

Tandemly repeated DNA is a major component of the human genome, and includes loci contributing to human disease. Minisatellites include the most variable human loci described to date, and the mechanisms by which this variation is generated in humans have been studied in detail. Integration of human minisatellites into yeast not only provides a model for further dissecting the molecular basis of length change mutation at these loci, but also more generally allows the study of complex recombinational events in yeast. We have used human minisatellite MS205 integrated into yeast to study the structural details of length change mutations. Apart from showing that mutation at this locus in yeast has features similar to those observed at some minisatellites in humans, including meiosis-specificity, and polarity, in which exchange events are localised to one extremity of the array, we here, for the first time, directly demonstrate that a flanking element in yeast regulates the mutation process. The results therefore support the hypothesis that flanking initiators are involved in minisatellite mutation in humans. Furthermore, mutant alleles showed more complex rearrangements in one orientation than the other. The data also suggest that the mutational pathway for deletions might be different from the pathway generating inter-allelic exchanges and duplications.

Induction of tissue transglutaminase in the trabecular meshwork by TGF-beta1 and TGF-beta2.

PURPOSE: To study whether human trabecular meshwork (HTM) cells are capable of expressing and secreting tissue transglutaminase (tTgase), an enzyme cross-linking extracellular matrix (ECM) proteins, and whether tTgase and synthesis of cross-linked fibronectin are increased after treatment of HTM cells with transforming growth factor (TGF)-beta1 or -beta2. METHODS: Anterior segments of six normal human eyes were stained with antibodies to tTgase. Tissues from three eyes were analyzed for tTgase using Western blot analysis. Monolayer cultures of HTM cells from eyes of five human donors were treated with 1.0 ng/ml TGF-beta1, -beta2, or 5 X 10(-7) M dexamethasone (DEX) for 12 to 96 hours. Induction of tTgase was investigated by Western and Northern blot analysis. External tTgase activity was measured by the ability to form polymerized fibronectin and the incorporation of biotinylated cadaverine into fibronectin. RESULTS: Labeling for tTgase was observed throughout the entire HTM. Cultured HTM cells expressed tTgase intra- and extracellularly. Treatment of cultured HTM cells with TGF-beta1 and -beta2 increased the tTgase mRNA and protein levels, whereas DEX had no effect. TGF-beta-treated HTM cells showed a significant increase in polymerized and unpolymerized fibronectin. Incorporation of biotinylated cadaverine was markedly increased when HTM cells were treated with TGF-beta for 24 hours before seeding. CONCLUSIONS: The enzyme tTgase is expressed in the HTM and is inducible by TGF-beta1 or -beta2 in cultured HTM cells. Extracellular tTgase is able to polymerize fibronectin. Increased levels of TGF-beta2 in the aqueous humor may lead to an increase of tTgase expression and activity in the HTM, causing an increase of irreversibly cross-linked ECM proteins. This mechanism might play a role for the increased outflow resistance seen in glaucomatous eyes.

Retinal vasculature changes in Norrie disease mice.

PURPOSE: To correlate morphologic changes in the retinal vasculature with degenerative changes in the neuronal retina of mice lacking 56 amino acids from the N-terminus of the Norrie disease (ND) gene product. METHODS: Posterior eye segments of ND mice of different age groups were investigated by light and electron microscopy (EM) and scanning EM of vascular corrosion cast preparations. The results were qualitatively and quantitatively compared with those obtained in age-matched littermate control mice and C57B1/6 mice. RESULTS: Quantitative evaluation revealed an increase in the number of blood vessels in the interface of the ganglion cell layer and the nerve fiber layer and a decrease in the inner and outer plexiform layers in ND mice older than 9 days compared with control mice. Vessels were also seen adjacent to the vitreous surface of the inner limiting membrane. Most of these vessels showed fenestrations and occasionally penetrated the inner limiting membrane. Hyaloid vessels were still present in the vitreous. The abnormal vascularization pattern was found in the entire retina and occurred in addition to the previously described alterations of the neuronal retina. CONCLUSIONS: There is a malformation of the retinal vasculature and a persistence of hyaloid vessels in the vitreous of ND mice. It is assumed that the ND gene product is required for normal vascularization of the inner retinal layers and for atrophy of hyaloid vessels.

AlphaB-crystallin in the trabecular meshwork is inducible by transforming growth factor-beta.

PURPOSE: Because in glaucomatous eyes transforming growth factor-beta (TGF-beta) and alphaB-crystallin are increased in the anterior eye segment, the effect of TGF-beta1 and TGF-beta2 on the expression of alphaB-crystallin and its corresponding mRNA was studied in human trabecular meshwork (TM) cells. METHODS: Monolayer cultures of "cribriform" and "corneoscleral" TM cells of 5 human donors (12-73 years of age) were treated with either 1.0 ng/ml TGF-beta1, TGF-alpha2, or 5 X 10(-7) dexamethasone (DEX) for 12 to 96 hours. Induction of alphaB-crystallin and the related mRNA was investigated by western and northern blot analyses. For comparison, human foreskin fibroblasts (HFF) and NIH 3T3 cells were treated in the same way as the TM cells. RESULTS: An increase of alphaB-crystallin mRNA was observed after treatment of TM cells with TGF-beta1 and TGF-beta2, whereas DEX had no effect. In the cribriform TM cells with a high basal level, the enhancement ranged between 2 and 3 times; whereas in the corneoscleral TM cells alphaB-crystallin mRNA increased between 5 and 6 times. Using western blot analysis, the increase of alphaB-crystallin expression in the cribriform TM cells was only small compared with the significant increase in the corneoscleral TM cells. Treatment of HFF and NIH 3T3 cells with TGF-beta did not induce alphaB-crystallin mRNA. CONCLUSIONS: This is the first time to show that alphaB-crystallin is not only induced by stress factors but also by TGF-beta in TM cell cultures. The difference in induction of mRNA and protein seems to be dependent on alphaB-crystallin concentration before treatment.

Evaluation of the rhodopsin knockout mouse as a model of pure cone function.

PURPOSE: To determine a time window in the rhodopsin knockout (Rho(-/-)) mouse during which retinal function is already sufficiently developed but cone degeneration is not yet substantial, thus representing an all-cone retina. METHODS: Electroretinograms (ERGs) were obtained from 14 homozygous Rho(-/-) mice and eight C57Bl/6 control mice. The same individuals were tested every 7 days, beginning as early as postnatal day (P)14. The ERG protocols included flash and flicker stimuli, both under photopic and scotopic conditions. Retinal and choroidal morphology was observed in animals of comparable age. RESULTS: Functionally, the developmental phase lasted until postnatal week (PW)3 in both the Rho(-/-) mice and the control animals. During PW4 to 6, the Rho(-/-) mice showed a plateau in ERG parameters with normal or even supernormal cone responses and complete absence of rod contributions. At PW7, there was a marked onset of degeneration, which progressed so that no ERG signals were left at PW13, when the control eyes still had normal ERG responses. Microscopically, cone degeneration paralleled the functional changes, beginning at approximately PW6 and almost complete at PW13, whereas retinal pigment epithelium (RPE) and choroid did not show any abnormalities. CONCLUSIONS: From PW4 to 6, Rho(-/-) mice appear to have normal cone and no rod function. Despite the missing rod outer segment (OS), the structure of retina, RPE, and choroid remained unchanged. Therefore, the Rho(-/-) mice can serve during this age period as a model for pure cone function. Such a model is particularly useful to evaluate rod-cone interaction and to dissect rod- from cone-mediated signaling pathways in vivo.

Localization of the stress proteins alpha B-crystallin and trabecular meshwork inducible glucocorticoid response protein in normal and glaucomatous trabecular meshwork.

PURPOSE: To examine the differential staining of two potential stress-response markers, alpha B-crystallin and the trabecular meshwork inducible glucocorticoid response protein (TIGR), in meshwork from normal and glaucomatous human eyes. METHODS: Trabecular meshwork from 35 eyes from 23 donors with either primary open-angle glaucoma, pseudoexfoliative glaucoma, or low-tension glaucoma, and from age-matched normal eyes, was examined. Sagittal and tangential frozen sections were stained with polyclonal antibodies to alpha B-crystallin or TIGR and then by a fluorescent secondary antibody. RESULTS: In normal eyes, labeling for alpha B-crystallin occurred in the subendothelial region of Schlemm's canal and outer corneoscleral regions, whereas TIGR labeling was found in the inner uveal meshwork region and the anterior portion of the meshwork. In contrast, in many glaucomatous eyes, labeling for alpha B-crystallin and TIGR occurred in more regions of the meshwork and appeared more intense than in normal eyes, regardless of the type or clinical severity of glaucoma. CONCLUSIONS: The differences in localization of alpha B-crystallin and TIGR may relate to functional specialization within meshwork tissues. The increase in the staining for these proteins in glaucomatous eyes could involve environmental and genetic factors in the disease processes.

Methyldopa does not alter the disposition of digoxin.

To investigate whether methyldopa alters digoxin disposition, eight healthy subjects received methyldopa titrated to 250 mg t.i.d. or placebo in a double-blind, cross-over manner for 16 consecutive days, with 0.25 mg intravenous digoxin coadministered on day 5 and 0.25 mg oral digoxin on days 9 to 16. Digoxin concentrations in plasma and urine were measured by RIA. Although assay sensitivity did not allow an adequate assessment of serum AUC(0-infinity) after intravenous administration, mean digoxin AUC(0-24) was 10.2 +/- 3.5 and 10.0 +/- 1.8 ng/ml X hr with placebo and methyldopa, respectively (P greater than 0.05). Mean urinary excretion after digoxin with or without methyldopa treatment was 0.204 +/- 0.34 and 0.197 +/- 0.38 mg, respectively. The mean steady-state serum concentrations of oral digoxin (AUC(0-24)/zeta) with and without methyldopa were 0.65 +/- 0.2 and 0.62 +/- 0.3 ng/ml, respectively. These data revealed no significant differences (P greater than 0.05) for various parameters with power of greater than 0.8 to detect meaningful differences of approximately 30 per cent. Thus, methyldopa did not alter digoxin disposition in healthy subjects, and a pharmacokinetic interaction in patients is unlikely.

Nitric oxide inhibits resting sphincter of Oddi activity.

The sphincter of Oddi has basal myogenic phasic activity that is modulated by neural and hormonal pathways. Stimulatory innervation to this organ is cholinergic, whereas the inhibitory pathways are unknown. Nitric oxide (NO), generated from L-arginine, relaxes gastrointestinal smooth muscle in vitro. We, therefore, hypothesized that resting sphincter of Oddi and duodenal motilities are regulated by a NO-mediated inhibitory pathway. In 23 anesthetized prairie dogs, systemic blood pressure and sphincter of Oddi and duodenal motilities were monitored during systemic infusion of N omega-nitro-L-arginine methyl ester (L-NAME), an inhibitor of nitric oxide synthase. L-NAME was infused alone and simultaneously with excess D- and L-arginine. L-NAME alone and L-NAME with D-arginine produced hypertension and increased sphincter of Oddi and duodenal motilities. L-arginine blocked these increases, suggesting that baseline sphincter of Oddi and duodenal motility regulation involves the generation of NO from L-arginine. We conclude that baseline sphincter of Oddi phasic activity is regulated by cholinergic stimulatory and NO-mediated inhibitory neural pathways.

Minisatellite mutation frequency in human sperm following radiotherapy.

Screening pedigrees for inherited minisatellite length changes provides an efficient means of monitoring repeat DNA instability but has given rise to apparently contradictory results regarding the effects of radiation on the human germline. To explore this further in individuals with known radiation doses and to potentially gain information on the timing of mutation induction, we have used an extremely sensitive single molecule approach to quantify the frequencies of mutation at the hypervariable minisatellites B6.7 and CEB1 in the sperm of three seminoma patients following hemipelvic radiotherapy. Scattered radiation doses to the testicles were monitored and pre-treatment sperm DNA was compared with sperm derived from irradiated pre-meiotic, meiotic and post-meiotic cells. We show no evidence for mutation induction in any of the patients and discuss this finding in the context of previous population studies using minisatellites as reporter systems, one of which provided evidence for radiation-induced germline mutation.

AlphaB-crystallin in lacrimal gland duct and tears.

PURPOSE: To investigate alphaB-Crystallin expression and localization in the lacrimal gland and tear fluid. METHODS: Mouse, rat, porcine, monkey and human lacrimal gland samples were immuno-histochemically and immuno-electron-microscopically stained with various antibodies against alphaB-crystallin. Western- and Northern-blotting was performed to demonstrate the presence of alphaB-crystallin mRNA and protein. Human tear fluid was analyzed for the presence of alphaB-crystallin using dot blotting. RESULTS: alphaB-Crystallin is located in the lacrimal gland duct cells but not in the acini. Electron microscopically, the protein was frequently found in apical electron-dense granules of lacrimal duct cells, occasionally also in the duct lumina. Western blotting confirmed the presence of alphaB-crystallin in the lacrimal gland, Northern blot samples revealed the presence of alphaB-crystallin mRNA. In the human tear fluid, alphaB-crystallin was present in all samples investigated. CONCLUSIONS: We demonstrate for the first time that alphaB-crystallin is present in the lacrimal gland. Presence of the protein in apical secretory granules as well as presence in the tear fluid might indicate secretion of alphaB-crystallin into the tear fluid.

Merkel cell distribution in the human eyelid.

Although Merkel cell carcinoma of the eye lid is reported frequently in the literature, only limited information exists about the distribution of Merkel cells in this tissue. Therefore, serial sections of 18 human cadaver eye lids (donors ages ranging between 63 and 97 years) were stained for cytokeratin 20 in various planes. The overall appearance of Merkel cells in these samples was low and mainly located in the outer root layer of the cilia hair follicles. Merkel cells were more frequent in the middle, and almost not detectable at the nasal and temporal edges. The localization is in accordance with that of Merkel cell carcinoma, but concerning the scarce appearance within this adulthood group, a specific physiological role of these cells in the eye lid is difficult to establish.

Age-dependent morphology of NADPH diaphorase-positive amacrine cells in the mouse retina.

NADPH diaphorase-positive amacrine cells (NAC) were studied in retinal whole mount preparation of mice, ranging from 1 day to 30 months of age. Following a peak in number and size during early development at postnatal day 14, their number and distribution remained well preserved up to senescence. Functional considerations include immunological, vascular and neuro-modulating aspects.

Staining and peeling of the internal limiting membrane using a fluorescent dye (Rhodamine 6 G).

AIM: To assess whether low concentrations of a fluorescent dye such as Rhodamine 6G would help the unaided human eye visualise the vitreous and the internal limiting membrane (ILM) under standard halogen illumination. MATERIAL/METHODS: The UV/Vis absorption (E) and fluorescence (I) spectra of Rhodamine 6G in water were measured and compared with Indocyanine Green (ICG). Surgery was performed in two rhesus monkeys and consisted of standard pars plana vitrectomy with halogen light source used for illumination. Rhodamine 6G was diluted in balanced salt solution (BSS). A few drops of the dye in a concentration of 0.1% (307 mOsm) were applied over the posterior pole in the air-filled globe and washed out by irrigation after 1 min. Immediately after surgery, the globes were enucleated, fixated and prepared for histological evaluation. RESULTS: In contrast to ICG, both the maximum of the absorption and emission of Rhodamin 6G are very much within the spectral sensitivity of the human eye. The Rhodamine 6G-BSS itself appears red in colour. Using a dye concentration of 0.1%, there was no visible red-staining of the ILM as such. As the dye was irrigated out with BSS, a marked green fluorescence of the fluid within the vitreous cavity was noted. With halogen illumination through a standard 20-gauge light pipe, the dye provided a sufficient green fluorescence to identify and safely remove the ILM and to clearly differentiate areas of peeled from non-peeled ILM. During light microscopy, eyes revealed a peeled ILM demarcation with no signs of acute retinal toxicity. CONCLUSION: The findings indicate that a fluorescent dye can be used for ILM peeling. Assuming that the fluorophore provides a high enough fluorescence quantum yield after adsorption to the ILM, much lower dye concentrations could be used compared with absorbent dyes, thereby minimising toxic effects.

An in vivo evaluation of Brilliant Blue G in animals and humans.

BACKGROUND/AIMS: To evaluate the retinal toxicity of Brilliant Blue G (BBG) following intravitreal injection in rat eyes and examine the biocompatibility and the staining properties in humans. METHODS: BBG was injected into the 11 rat eyes to evaluate toxic effects with balanced salt solution (BSS) serving as control. Retinal toxicity was assessed by retinal ganglion cell (RGC) counts and by light microscopy 7 days later. In addition, BBG was applied during vitrectomy for macular hole (MH) (n = 15) or epiretinal membranes (ERM) (n = 3) in a prospective, non-comparative consecutive series of patients. Before and after surgery, all patients underwent a complete clinical examination including measurement of best corrected visual acuity (VA) and intraocular pressure, perimetry, fundus photography and optical coherence tomography. Patients were seen 1 day before surgery and then in approximately four weeks intervals. RESULTS: No significant reduction in RGC numbers and no morphological alterations were noted. A sufficient staining of the internal limiting membrane (ILM) was seen in patients with MH, while the staining pattern in ERM cases was patchy, indicating that parts of the ILM were peeled off along with the ERM in a variable extent. All MHs could be closed successfully. VA improved in 10 eyes (56%; 8/15 MH patients, 2/3 ERM patients), was unchanged in four eyes (22%; all MH patients) and was reduced in four eyes (22%; 3/15 MH, 1/3 ERM). No toxic effects attributable to the dye were noted during patient follow-up. The ultrastructure of tissue harvested during surgery was unremarkable. CONCLUSION: Brilliant Blue provides a sufficient and selective staining of the ILM. No retinal toxicity or adverse effects related to the dye were observed in animal and human studies. The long-term safety of this novel dye will have to be evaluated in larger patient series and a longer follow-up.

Proteins with an endostatin-like domain in a mouse model of oxygen-induced retinopathy.

Neovascularization in the retinopathy of prematurity (ROP) mouse eye is a self-limiting phenomenon. Free endostatin is known to be anti-angiogenic. In this study, we identified the localization of endostatin-like protein (ELP) sequences and investigated their possible role in this process. ROP was induced in C57Bl/6 mice and the eyes observed 1-11 days after termination of high oxygen supply (P13-P21). Sagittal sections and retinal flatmounts were double-stained with antibodies against a protein-sequence of endostatin, vascular endothelial growth factor (VEGF), lectin, and smooth-muscle alpha actin. The fluorescence was visualized by traditional and confocal microscopy. Intense staining for VEGF in the inner retina was limited to the early stages of neovascularization and diminished at P19-P21. In contrast, staining for ELPs appeared at P15 around the newly formed vessels and remained even after degeneration of their endothelial cells. Staining of the inner retinal vasculature for ELPs was restricted to P17-P19, the known maximum of the neovascular response. Outer retinal vessels did not show presence of ELPs at any time. Our study demonstrates that ELPs, absent at the beginning of neovascular sprouting, increases with the amount of neovascularization and thus, varies reciprocally to VEGF in the time period investigated. ELPs remain during the regression of the vessels and might therefore play an important role in the self-limiting process of ROP neovascularization.

Description and function of the ciliary nerves--some historical remarks on choroidal innervation.

The earliest accounts of the eye recognized its main function as providing vision, but the mechanism of how the eye functioned remained obscure for many centuries. The aim of the following work is to outline these changes in the understanding of a particular structure and function of the human body, namely, the ciliary nerves supplying the uveoscleral part of the eye. In the extensive study on the history of ophthalmology by Hirschberg (published between 1899 and 1918), the ciliary nerves and choroidal innervation are only sparsely mentioned.