Analysis of progressive ophthalmic lesion in a patient with subacute sclerosing panencephalitis.

PURPOSE: To evaluate the progressive lesions affecting the visual system in a patient with subacute sclerosing panencephalitis (SSPE). METHODS: The authors observed a 15-year-old boy with SSPE. Since the diagnosis was made before the appearance of ocular manifestations, the authors recorded the progressive ocular lesions using various ophthalmic examinations. RESULTS: The patient showed no ophthalmic abnormalities until he developed a left homonymous hemianopia with sudden bilateral disturbed visual acuity. Severe progressive macular lesions including a pigment epithelial window defect by fluorescein angiography, a marked decrease in foveal thickness by optical coherence tomography, and an extensive disorder mainly specific to cone cells in the central retina by electroretinography were demonstrated. Novel findings such as a transient relative afferent pupillary defect and an anterior uveitis were also observed. CONCLUSIONS: Analyses over a long period of time showed progressive ophthalmic findings in a patient with SSPE.

Isolation of Mycoplasmas from nasal swabs of calves affected with respiratory diseases and antimicrobial susceptibility of their isolates.

Nasal swabs of 293 calves were examined for Mycoplasma. The samples were collected from calves affected with respiratory diseases on 71 farms in various parts of Japan between 1996 and 1997. Mycoplasma bovirhinis was isolated from 47 of 293 calves (16.0%). Mycoplasma alkalescens, M. bovis, M. arginini, M. bovigenitalium and Acholeplasma spp. were isolated from 19 (6.5%), seven (2.4%), four (1.4%), four (1.4%) and 18 (6.1%) calves, respectively. Pasteurella multocida and P. haemolytica were isolated from 60% of Mycoplasma-positive calves. However, other bacteria were not isolated from calves. To evaluate the antimicrobial susceptibility of their isolates, 68 M. bovirhinis, 21 M. alkalescens and 10 M. bovis strains were examined for 12 antimicrobial agents. All isolates showed higher susceptibility to tiamulin than to the other drugs used in the study. However, erythromycin had no effect on any of the Mycoplasma strains studied. The field isolates were less susceptible than the type strains to some drugs, such as spiramycin, oxytetracycline and tylosin.

Structural characterization of heparan sulfate and chondroitin sulfate of syndecan-1 purified from normal murine mammary gland epithelial cells. Common phosphorylation of xylose and differential sulfation of galactose in the protein linkage region tetrasaccharide sequence.

Syndecan-1, present on the surfaces of normal murine mammary gland epithelial cells, is a transmembrane hybrid proteoglycan, which bears glycosaminoglycan (GAG) side chains of heparan sulfate (HS) and chondroitin sulfate (CS). Purified syndecan-1 ectodomains were analyzed for disaccharide composition and the GAG-protein linkage region after digestion with bacterial lyases. The HS chains contained predominantly a nonsulfated unit with smaller proportions of two monosulfated, two disulfated, and a trisulfated unit, whereas CS chains were demonstrated for the first time to bear GlcUA-GalNAc(4-O-sulfate) as a major component as well as GlcUA-GalNAc, GlcUA-GalNAc(6-O-sulfate), and an E disaccharide unit GlcUA-GalNAc(4,6-O-disulfate) as minor yet appreciable components. Two kinds of linkage region tetrasaccharides, GlcUA-Gal-Gal-Xyl and GlcUA-Gal-Gal-Xyl(2-O-phosphate), were found for the HS chains in a molar ratio of 55:45. In marked contrast, an additional sulfated tetrasaccharide, GlcUA-Gal(4-O-sulfate)-Gal-Xyl, was demonstrated only for the CS chains, and the unmodified phosphorylated and sulfated components were present at a molar ratio of 55:26:19. The present study thus provided conclusive evidence for the hypothesis that 4-O-sulfation of Gal is peculiar to CS chains in contrast to the phosphorylation of Xyl, which is common to both HS and CS chains. These modifications may be required for biosynthetic maturation of the linkage region tetrasaccharide sequence, which is a prerequisite for creating the repeating disaccharide region of GAG chains and/or biosynthetic selective chain assembly of CS and HS chains.

Functions of cell surface heparan sulfate proteoglycans.

The heparan sulfate on the surface of all adherent cells modulates the actions of a large number of extracellular ligands. Members of both cell surface heparan sulfate proteoglycan families, the transmembrane syndecans and the glycosylphosphoinositide-linked glypicans, bind these ligands and enhance formation of their receptor-signaling complexes. These heparan sulfate proteoglycans also immobilize and regulate the turnover of ligands that act at the cell surface. The extracellular domains of these proteoglycans can be shed from the cell surface, generating soluble heparan sulfate proteoglycans that can inhibit interactions at the cell surface. Recent analyses of genetic defects in Drosophila melanogaster, mice, and humans confirm most of these activities in vivo and identify additional processes that involve cell surface heparan sulfate proteoglycans. This chapter focuses on the mechanisms underlying these activities and on the cellular functions that they regulate.

The gene structure and promoter sequence of mouse hyaluronan synthase 1.

The structure and organization of mouse hyaluronan synthase 1 gene, HAS1 were determined by direct sequencing of lambda phage clones carrying the entire gene and by application of the long and accurate (LA)-PCR method to amplify regions encompassing the exon-intron boundaries and all of the exons. This gene spans about 11kb of genomic DNA and consists of 5 exons and 4 introns. A similarity in the exon-intron organization was found between the genes of mouse HAS1 and Xenopus laevis DG42 which was recently identified as Xenopus hyaluronan synthase. The transcription initiation site was determined by rapid amplification of the cDNA ends (5'-RACE). Position +1 is located 55 nucleotides upstream of the ATG initiation codon. The promoter region of the HAS1 gene has no typical TATA box, but contains a CCAAT box located 190 nucleotides upstream of the transcription initiation site. Further analysis of 1.4 kb of the 5' flanking region revealed several potential binding motifs for transcription factors. This information about the gene structure may be useful for further studies on the promoter activity.

The gene structure and organization of mouse PG-Lb, a small chondroitin/dermatan sulphate proteoglycan.

PG-Lb was originally characterized as a small chondroitin/dermatan sulphate proteoglycan expressed preferentially in the zones of flattened chondrocytes in developing chick limb cartilage. The occurrence of this proteoglycan in mammalian cartilage has been shown by the isolation of a cDNA clone from mouse cartilage cDNA library [Kurita, Shinomura,Ujita, Zako, Kida, Iwata and Kimata (1996) Biochem. J. 318, 909-914]. To understand the regulation mechanisms for such a unique expression, we have investigated a genomic DNA structure of the PG-Lb gene. The gene is composed of seven exons and six introns spanning more than 50 kb. The leucine-rich repeats are encoded from exon V to exon VII. The transcription initiation site has been determined by rapid amplification of the cDNA ends ('5'-RACE'). The possible TATA box was detected about 90 bp upstream of the adenosine residue that was numbered as position +1. Further analyses of 1.5 kb of the 5' flanking region and 2.2 kb of the first intron have revealed several potential binding motifs for transcription factors such as Sox 5 and 9. The presence of those sequences in the PG-Lb gene was discussed in relation to the unique expression of this proteoglycan. The chromosomal localization of the murine PG-Lb gene was determined to be on the mouse chromosome 10 by the fluorescence-in-situ-hybridization ('FISH') method.

Alternative splicing of the unique "PLUS" domain of chicken PG-M/versican is developmentally regulated.

We investigated the occurrence of alternatively spliced forms (V0, V1, V2, and V3) of PG-M/versican, a large chondroitin sulfate proteoglycan in developing chicken retinas, using the reverse transcription-polymerase chain reaction. We characterized the PLUS domain, which is apparently unique to the chicken molecule and is regulated by alternative splicing. PG-M in chicken retinas consisted of four forms with (V0, V1, V2, and V3) and two forms without (V1 and V3) the PLUS domain (PG-M+ and PG-M-, respectively). The four forms of PG-M+ were found in all samples examined, but the occurrence of the two PG-M- forms was regulated developmentally. Genomic analysis has revealed that the PLUS and CS-alpha domains are encoded by a single exon, and this exon has an internal alternative 5'-splice donor site, allowing alternative spliced forms that do not include the 3'-end of the exon. Sequences corresponding to the chicken PLUS domain (plus) were not found in mouse and human and may have disappeared during evolution. Sequence similarity suggests that the PLUS domain corresponds to the keratan sulfate attachment domain of aggrecan and that it has a distinct function in the chicken eye.

Transient expression of PG-M/versican, a large chondroitin sulfate proteoglycan in developing chicken retina.

We previously showed the expression of PG-M/versican in embryonic chicken retina. In this study, we characterized the alternatively spliced forms of PG-M/versican and their developmental regulation to investigate the implication of PG-M/versican in neurite outgrowth from retinal cells during development. On day 5, the immunolocalization of PG-M was first observed at the inner surface of neural retina. On day 7, the pronounced staining was observed in the nerve fiber layer and inner plexiform layer where neural networks of ganglion cells were being formed. As the development proceeded, more intensive staining was observed in these layers. The staining peaked on day 14 and then decreased. Northern analysis and western blotting revealed the presence of a single-sized transcript (13 kb) and the PG-M/versican core protein (550 kDa) on day 14, but the absence of any transcripts or protein bands on day 20, indicating a transient expression of PG-M+ (VO), the alternatively spliced form with the most abundant sites for the chondroitin sulfate attachment. Taken together, it is likely that PG-M/versican is involved in neurite outgrowth from ganglion cells during retinal development, and antiadhesion activity of its chondroitin sulfate chains may be important for regulation.

Occurrence of PG-Lb, a leucine-rich small chondroitin/dermatan sulphate proteoglycan in mammalian epiphyseal cartilage: molecular cloning and sequence analysis of the mouse cDNA.

PG-Lb is a chondroitin/dermatan sulphate proteoglycan first isolated from chick embryo limb cartilage. It had been assumed that osteoglycin represents its mammalian homologue. However, partial amino acid sequences of a novel proteoglycan from bovine epiphyseal cartilage showed high identity with those of chick PG-Lb (P. Neame, L. Rosenberg and M. Höök, personal communication). Reverse transcriptase PCR using degenerate oligonucleotide primers gave a cDNA fragment that might correspond to mouse PG-Lb. We isolated a clone from a cDNA library of newborn mouse epiphyseal cartilage using the cDNA fragment as a probe. The cloned cDNA was 1430 bp long and contained a 966 bp open reading frame which encoded the core protein consisting of 322 amino acid residues. The deduced amino acid sequence showed a high overall identity with chick PG-Lb (about 62%, reaching about 80% over the carboxyl two-thirds). In addition, the amino acid sequence contained a signal peptide, six cysteine residues at the invariant relative position to chick PG-Lb, six leucine-rich repeats at the carboxyl two-thirds, three possible glycosaminoglycan-attachment sites (two sites at the N-terminal side and one site at the C-terminus) and two possible Asn-glycosylation sites near the C-terminus. Northernblot analysis demonstrated the specific expression of a 1.5 kb message in cartilage and testis. These structural features and the characteristic expression suggest that the cloned molecule is mouse PG-Lb.

The gene structure and organization of mouse PG-M, a large chondroitin sulfate proteoglycan. Genomic background for the generation of multiple PG-M transcripts.

We previously showed not only the presence of multiple RNA transcripts of different sizes encoding the core protein of mouse PG-M, but also their tissue-dependent expression. Major causes for the multiple forms were found to be due to alternative usage of the two different chondroitin sulfate attachment domains (alpha and beta). In this study, genomic DNA analysis has revealed that these domains are encoded by two large exons, exon VII (2880 base pairs) and exon VIII (5229 base pairs). The splice sites of these two exons were consistent with the occurrence of alternative splicing without frameshift. Furthermore, the mouse PG-M gene was shown to have four distinct polyadenylation signals and three candidates for the transcription initiation site as well. These genomic structural variations may contribute to the multiplicity of PG-M transcripts. Northern hybridization analysis showed that at least three different transcripts were generated by different usage of the distinct polyadenylation signals.

Expression of PG-M(V3), an alternatively spliced form of PG-M without a chondroitin sulfate attachment in region in mouse and human tissues.

We showed previously that the alternative splicing of chondroitin sulfate attachment domains (CS alpha and CS beta) yielded multiforms of the PG-M core protein in mouse. A transcript encoding a new short form of the core protein PG-M(V3) was found in various mouse tissues using polymerase chain reaction. DNA sequences of the polymerase chain reaction products suggested that PG-M(V3) had no chondroitin sulfate attachment domain. PG-M(V3) was also detected in various human tissues. The presence of a transcript for PG-M(V3) was further supported by Northern blot analysis. Southern blot analysis confirmed that multiforms of the PG-M core protein, including PG-M(V3), were derived from a single genomic locus by an alternative splicing mechanism. Because PG-M(V3) has no chondroitin sulfate attachment region, which is the most distinctive portion of a proteoglycan molecule, this form may have a unique function.

Multiple forms of mouse PG-M, a large chondroitin sulfate proteoglycan generated by alternative splicing.

We have isolated and sequenced cDNA clones that encode the core protein of PG-M-like proteoglycan produced by cultured mouse aortic endothelial cells (Morita, H., Takeuchi, T., Suzuki, S., Maeda, K., Yamada, K., Eguchi, G., and Kimata, K. (1990) Biochem. J. 265, 61-68). A homology search of the cDNA sequence has suggested that the core protein is a mouse equivalent of chick PG-M(V1), one of the alternatively spliced forms of the PG-M core protein, which may correspond to human versican. Northern blot analysis revealed three mRNA species of 10, 9, and 8 kilobases (kb) in size. The analysis of PG-M mRNA species in embryonic limb buds and adult brain revealed the presence of other mRNA species with different sizes; the one with the largest size (12 kb) was found in embryonic limb buds, and the ones with smaller sizes of 7.5 and 6.5 kb were in adult brain. Sequencing of cDNA clones for the smaller forms in the adult brain showed that they were different from PG-M(V1) in encoding the second chondroitin sulfate attachment domain (CS alpha) alone. Occurrence of the PCR products striding over the junction of the first and second chondroitin sulfate attachment domains suggested that a mRNA of 12 kb in size corresponded to a transcript without the alternative splicing (PG-M(V0)). It is likely, therefore, that multiforms of the PG-M core protein may be generated by alternative usage of either or both of the two different chondroitin sulfate attachment domains (alpha and beta) and that molecular forms of PG-M may vary from tissue to tissue by such an alternative splicing.

Anterior chamber depth after posterior chamber intraocular lens implantation.

The precise behavior of the anterior chamber depth after intraocular lens implantation is uncertain. To ascertain variations in the values at different time points, we measured the anterior chamber depth in 38 eyes that had undergone extracapsular cataract extraction and posterior chamber intraocular lens implantation. Measurements were performed by ultrasonography preoperatively and at 2 days, 1 and 2 weeks postoperatively, and at 1-month intervals thereafter up to 10 months (mean follow-up period, 9 months). Results showed that two peaks of the anterior chamber depth occurred at 1 week and 3 months postoperatively (means +/- SD, 3.54 +/- 0.39 and 3.59 +/- 0.43 mm, respectively). A significant difference was found between the values measured preoperatively (3.29 +/- 0.57) and at 2 days postoperatively (3.33 +/- 0.39) and the 1-week postoperative value (p < 0.05, using the one-way analysis of variance test). A significant difference also was found between the preoperative and the monthly measurements and the 3-month postoperative measurement (p < 0.05). We also calculated the refractive error and found that during the second postoperative week, the refraction tended toward higher myopia that became more hyperopic 3 months postoperatively. This anterior chamber depth variation may cause variations in vision after cataract surgery with intraocular lens implantation.