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Biomedical subjects

J A Guggenheim

Publications and source records attributed to J A Guggenheim.

11 recordsLinked to original sources
PubMed2005

Gelatinous drop-like corneal dystrophy in a child with developmental delay: clinicopathological features and exclusion of the M1S1 gene.

AIMS: Gelatinous drop-like corneal dystrophy (GDLD) is an early-onset, autosomal recessive condition characterised by amyloid deposits within the cornea. We report the histopathological and molecular genetic findings in a Caucasian child with GDLD who also exhibited global developmental delay. METHODS: Bilateral lamellar keratoplasty was carried out at age 6 and 7 years. Tissue was fixed for light and electron microscopy, including immunoelectronmicroscopy. The coding region of the M1S1 gene was screened for mutations in the affected proband and available relatives, using DNA extracted from mouthwashes. RESULTS: Nodular deposits, which were present subepithelially and in the central superficial stroma, stained typically for amyloid with PAS and Congo red. A nodular deposit of amyloid, together with large amounts of lactoferrin and sparse amounts of keratoepithelin (betaig-h3), was present in the central superficial stroma, causing destruction of Bowman's layer and elevation of the thinned, degenerate epithelium. Around the deposit zone, the stroma exhibited large numbers of thick filamentous proteoglycan deposits. While the affected child was homozygous for a novel A1133 C single-nucleotide polymorphism (SNP) that resulted in an aspartic acid to alanine substitution at position 173 of the M1S1 coding sequence, this polymorphism was also found at relatively high frequency in a sample of normal controls, enabling exclusion of the M1S1 gene as the disease locus. CONCLUSION: Increased epithelial permeability in GDLD may be explained in part by an altered membrane permeability of the superficial epithelial cells. An association with developmental delay has not been reported previously.

Amyloid↗
PubMed2003

Myopia, genetics, and ambient lighting at night in a UK sample.

BACKGROUND: It has been reported that exposure to artificial lighting at night during the first 2 years of life was very strongly associated with subsequent myopia development. METHODS: The strength of this association was tested in a UK sample for the first time. The study population comprised 122 university students. RESULTS: Myopia occurred with approximately equal frequency in those who slept with and without light exposure at night. In contrast, two largely genetic factors, parental myopia and race, were both significantly associated with myopia development, as has been found previously. CONCLUSION: This study provides further support for the view that night-time light exposure during infancy is not a major risk factor for myopia development in most population groups. In a subset of this cohort for which spectacle prescriptions were available for both parents (49 trios), the heritability of ocular refraction was estimated to be 0.31.

Adult↗
PubMed1999

Prevention of depurination during elution facilitates the reamplification of DNA from differential display gels.

DNA fragments that show a pattern of differential expression on differential display gels must be eluted from the gel matrix and reamplified to enable further analysis. Elution is usually achieved by heating excised gel slices in a small volume of either water or TE. Here we show that this elution step can adversely affect the ability of the eluted DNA to act as a template for PCR reamplification, probably via the process of depurination. Simply switching to an elution solvent designed to minimise depurination (PCR buffer) facilitates the elution of intact DNA fragments. This improvement is likely to be most beneficial when eluting higher molecular weight fragments (e.g. those >500 bp), in situations where the amount of DNA in an excised gel slice is limited or when contaminating differential display products co-migrate with the differentially expressed product.

DNA↗
PubMed1999

Mammalian polyadenylation sites: implications for differential display.

Differential display relies on a series of anchored primers to divide the total mRNA population into subsets of roughly equal size. However, this will only occur if the dinucleotide targeted by the anchor region of the anchored primers has a random frequency distribution [i.e. each of the 12 possible dinucleotides preceding the poly(A) tail occur with the same frequency]. Previous reports have suggested that this is not the case and that the frequency distribution of the targeted dinucleotide can vary as much as 10-fold. In an analysis of several hundred unrelated mammalian mRNA sequences, we confirmed that the frequency of this particular dinucleotide does vary, although <3-fold. Of equal importance, however, we found that the number of bands displayed with each of the respective anchored primers was not affected by these variations in dinucleotide frequency, suggesting that anchored primer promiscuity permits mispriming during the reverse transcription stage of differential display. Close examination of this issue suggested that both mispriming at the anchor region and internal mispriming are common in differential display reverse transcription and implies that repetitive sampling occurs exten-sively in differential display. Thus, reverse transcriptase mispriming may considerably reduce the efficiency of differential display.

Animals↗
PubMed1996

Form-deprivation myopia induces activation of scleral matrix metalloproteinase-2 in tree shrew.

PURPOSE: To investigate whether structural changes to the sclera during form-deprivation myopia are caused by active tissue remodeling, the gelatinase activity of tree shrew scleras was studied in normal animals, form-vision deprived animals, and animals recovering from myopia. METHODS: Infant tree shrews were monocularly deprived (MD) of form vision with translucent occluders for 5 days. Recovery animals were allowed 3 days of binocularly unoccluded vision after the period of form deprivation. Eyes were removed and dissected to provide scleral samples corresponding to equatorial and posterior regions. Gelatinase activity was assessed by quantitative sodium dodecyl sulfate-polyacrylamide gel electrophoresis or SDS-PAGE, gelatin, zymography of scleral matrix metalloproteinase (MMP) extracts. RESULTS: The major gelatinolytic species present in tree shrew sclera was found to be MMP-2 (gelatinase A). In normal (nondeprived) animals, most of the MMP-2 was found to be in the latent form (the ratio of active-to-latent MMP-2 was 0.23 +/- 0.05 and 0.34 +/- 0.06 in equatorial and posterior samples, respectively; n = 10 eyes from five animals). After 5 days of MD, there was a threefold increase in the amount of active scleral MMP-2 in myopic eyes compared to contralateral control eyes, whereas latent MMP-2 activity levels were not altered significantly. This increase in active MMP-2 was seen in both the equatorial and posterior sclera of myopic eyes (active-to-latent MMP-2 ratios were 0.53 +/- 0.10 and 0.81 +/- 0.09 in equatorial and posterior regions, respectively; n = 6 animals). Contralateral control eyes had levels of both active and latent MMP-2 not significantly different from normal eyes. After only 3 days of unoccluded vision, previously deprived eyes that were now recovering from myopia had a fivefold lower level of active MMP-2 than that seen in deprived eyes after 5 days of MD. In fact, active (and latent) MMP-2 levels were reduced in recovering eyes even below the levels found in their contralateral control eyes. Active-to-latent ratios for recovering eyes were 0.11 +/- 0.03 and 0.15 +/- 0.03 in equatorial and posterior sclera, respectively (n = 5 animals). CONCLUSIONS: These results demonstrate that form-deprivation myopia and recovery from myopia alter scleral catabolism and provide further support for the theory that changes in eye size during mammalian refractive development are the result of active tissue remodeling rather than passive scleral stretching alone.

Animals↗
PubMed1995

Chloride binding in the stroma of cultured human corneas.

In the ox cornea, more than half of the non-diffusible, matrix negative charge is derived from the binding of free chloride ions. Because the magnitude of the net matrix charge is the dominant factor which determines the degree of stromal swelling, we investigated whether this phenomenon, stromal chloride binding, also occurs in human corneal stroma. Intrastromal ion concentrations were measured with radio-isotopes when human (outdated Eye Bank) corneas or (fresh) bovine corneas, physically clamped to maintain a constant hydration, were incubated in buffered 154 mM NaCl. The intrastromal chloride ion concentration was compared to the normalized concentrations of trace quantities of radio-labelled acetate and lactate ions. For human corneas, the intrastromal chloride ion concentration was found to be significantly higher (P < 0.001, t-test) than the normalized concentrations of both acetate and lactate ([Cl]i = 142.5 +/- 0.9 mM, (n = 9); [acetate]i = 131.2 +/- 1.2 mM, (n = 8); [lactate]i = 131.9 +/- 1.5 mM, (n = 5); all values are mean +/- S.E.M.). The sodium ion concentration was elevated ([Na]i = 176.0 +/- 1.8 mM, (n = 9)). These results demonstrate that chloride binding occurs to a significant extent in cultured human corneal stroma and suggest that chloride binding may be evident in the native human cornea.

Animals↗
PubMed1994

Localization of Na+/K(+)-ATPase in the bovine corneal endothelium.

A mouse monoclonal antibody has been used to localize Na+/K(+)-ATPase in the bovine corneal endothelium. The specificity of the antibody was demonstrated by reaction with a single protein of molecular mass 100 kDa on Western blots and immunoprecipitation of a complex consisting of 100 kDa and 50 kDa subunits. Treatment of the immunoprecipitated antigen with Peptide N-Glycanase F produced no change in the molecular mass of the 100 kDa protein, but resulted in a progressive decrease in the molecular mass of the 50 kDa subunit, to yield a core protein of molecular mass about 33 kDa. The pattern of deglycosylation suggested the presence of three N-linked glycans attached to the 33 kDa protein core. These results were consistent with the antibody being specific for the alpha subunit of the Na+/K(+)-ATPase. Immunocytochemical studies at the light and electron microscopic level demonstrated antibody binding to both the basal and lateral membranes of bovine corneal endothelial cells. This suggested a baso-lateral distribution of Na+/K(+)-ATPase in these cells, rather than the previously proposed lateral membrane-only distribution.

Animals↗
PubMed1994

Sodium movement into and out of corneal endothelium.

Rabbit corneal endothelial cells mounted in vitro were impaled simultaneously with Na(+)-selective and conventional KCl-filled microelectrodes. The membrane potential (Vm) was -30.4 +/- 0.8 mV (mean +/- SEM, n = 55) and the intracellular [Na+]i (calculated from the Na(+)-selective electrode potential, VNa) was 13.7 +/- 1.9 mM (mean +/- SEM, n = 16). When ouabain was added to the perfusate the cell depolarised, causing both Vm and VNa to increase with a very similar time course. Final Vm was -6.3 +/- 0.6 mV (mean +/- SEM, n = 15), and the final [Na+]i was 114 +/- 6.9 mM (mean +/- SEM, n = 5). The parallel increase in Vm and rise in [Na+]i suggest that a component of the ouabain-induced depolarisation of the cell (increase in Vm) is due to Na+ entry into the cell down its concentration gradient. The lateral and basal location of the Na+/K(+)-ATPase in bovine endothelial cells was confirmed (for the first time at the electron-microscopic level) using a monoclonal antibody specific for the alpha 1 subunit of Na+/K(+)-ATPase. The absence of a net Na+ flux across these cells combined with the basolateral location of the ATPase suggest that Na+ exit from the cell, and its re-entry take place across the same membrane (i. e. the basolateral).

Animals↗