Controlling joint instability after anterior cruciate ligament transection inhibits transforming growth factor-beta-mediated osteophyte formation.

OBJECTIVE: Abnormal joint instability contributes to cartilage damage and osteophyte formation. We investigated whether controlling joint instability inhibited chronic synovial membrane inflammation and delayed osteophyte formation and examined the role of transforming growth factor-beta (TGF-ß) signaling in the associated mechanism. DESIGN: Rats (n = 94) underwent anterior cruciate ligament (ACL) transection. Anterior tibial instability was either controlled (CAM group) or allowed to continue (SHAM group). At 2, 4, and 8 weeks after surgery, radiologic, histopathologic, immunohistochemical, immunofluorescent, and enzyme-linked immunosorbent assay examinations were performed to evaluate osteophyte formation and TGF-ß signaling. RESULTS: Joint instability increased cartilage degeneration score and osteophyte formation, and cell hyperplasia and proliferation and synovial thickening were observed in the synovial membrane. Major findings were increased TGF-ß expression and Smad2/3 following TGF-ß phosphorylation in synovial membarene, articular cartilage, and the posterior tibial growth plate (TGF-ß expression using ELISA: 4 weeks; P = 0.009, 95% CI [260.1-1340.0]) (p-Smad2/3 expression density: 4 weeks; P = 0.024, 95% CI [1.67-18.27], 8 weeks; P = 0.034, 95% CI [1.25-25.34]). However, bone morphogenetic protein (BMP)-2 and Smad1/5/8 levels were not difference between the SHAM model and the CAM model. CONCLUSIONS: This study showed that the difference between anterior tibial instability caused a change in the expression level of TGF in the posterior tibia and synovial membrane, and the reaction might be consequently involved in osteophyte formation.

Capture probability of released males of two Bactrocera species (Diptera: Tephritidae) in detection traps in California.

The genus Bactrocera (Diptera: Tephritidae) includes approximately 70 polyphagous species that are major pests of fruit and vegetable crops. Most Bactrocera species have limited geographic distributions, but several species are invasive, and many countries operate continuous trapping programs to detect infestations. In the United States, California maintains approximately 25,000 traps (baited with male lures) specifically for Bactrocera detection distributed over an area of approximately 6,400 km2 (2,500 miles2) in the Los Angeles area. Although prior studies have used male lures to describe movement of Bactrocera males, they do not explicitly relate capture probability with fly distance from lure-baited traps; consequently, they do not address the relative effectiveness of male lures in detecting incipient populations of Bactrocera species. The objective of this study was to measure the distance-dependent capture probability of marked, released males of Bactrocera dorsalis (Hendel) and Bactrocera cucurbitae (Coquillett) (methyl eugenol- and cue lure-responding species, respectively) within the detection trapping grid operating in southern California. These data were then used to compute simple probability estimates for detecting populations of different sizes of the two species. Methyl eugenol was the more powerful attractant, and based on the mark-recapture data, we estimated that B. dorsalis populations with as few as approximately 50 males would always (>99.9%) be detected using the current trap density of five methyl eugenol-baited traps per 2.6 km2 (1 mile2). By contrast, we estimated that certain detection of B. cucurbitae populations would not occur until these contained approximately 350 males. The implications of the results for the California trapping system are discussed, and the findings are compared with mark-release-recapture data obtained for the same two species in Hawaii.

Reversal of intraocular pressure increases with 0.5% apraclonidine after dilated fundus examination in patients with chronic open-angle glaucoma.

BACKGROUND: Apraclonidine 1.0% has been shown to reverse the potential intraocular pressure (IOP) increase after pupil dilation IOP increases in patients with chronic open-angle glaucoma. However, it is only approved for preventing IOP spikes after laser surgery. The purpose of this study is to determine the effectiveness of 0.5% apraclonidine in reversing IOP increases after pupillary dilation in patients with chronic open-angle glaucoma. METHODS: Twenty-two patients with chronic open-angle glaucoma were found to have an increase in post-dilation IOP of at least 4 mmHg from pre-dilated levels (baseline) in both eyes. IOP was measured 1 hour after dilation, after which two drops of 0.5% apraclonidine were instilled in one eye and the IOP was remeasured 15 minutes later in both eyes. Instillation of 0.5% apraclonidine in one eye was continued every 15 minutes and IOP was measured 15 minutes after each instillation, until the pressure returned to baseline levels. RESULTS: The IOP of the initially treated eye of all 22 patients returned to within levels clinically insignificant from baseline IOP within 90 minutes. By comparison, the IOP of the control group (untreated eye) remained elevated. Once the initial treatment eye returned to baseline levels, the control group was then treated with 0.5% apraclonidine, resulting in a lowering effect of the IOP in similar fashion to the initial treated group. CONCLUSIONS: Apraclonidine 0.5% appears to be effective in reduction of post-dilated IOP increases in patients with chronic open-angle glaucoma.

Spectrophotometric determination of lithium ion using a water-soluble octabromoporphyrin in aqueous solution.

A water-soluble porphyrin, (2,3,7,8,12,13,17,18-octabromo-5,10,15,20-tetrakis(4-sulfonatophenyl)porphyrin; H(2)obtpps(4-)) was synthesized and developed for the determination of lithium ion in aqueous solution. The octabromo groups lower the basicity of the porphyrin by their electron-withdrawing effect, and enable the porphyrin to react with the lithium ion in alkaline solution to form the lithium complex along with a shift of absorption maximum: lambda max/nm (logepsilon/mol(-1) dm(3) cm(-1)) of the lithium porphyrin are 490.5 nm (5.31) and 734 nm (4.36). Sodium and potassium ions did not react with the porphyrin. The equilibrium constant for the reaction Li(+)+Hobtpps(5-)right harpoon over left harpoon[Li(obtpps)](5-)+H(+) was found to be 10(-8.80) and the conditional formation constant of the [Li(obtpps)](5-) at pH 13 is 10(4.21). The above results were applied to the determination of lithium ion in aqueous solution. The interference from transition and heavy metal ions was masked by using N,N'-1,2-ethanediylbis[N(carboxylmethy)glycinato]magnesium(II) ([Mg(edta)](2-)) solution. Absorbance at 490 nm was measured against a blank solution. A calibration graph was linear over the range of 0.007-0.7 mug cm(-3) (1x10(-6)-1x10(-4) mol dm(-3)) of lithium(I) with a correlation factor of 0.967. Lithium ion less than ppm level was determined spectrophtometrically in aqueous solution. The proposed method was applied to the determination of lithium in human serum and sea water samples.

Eye injury from tarantula.

BACKGROUND: Tarantulas have become increasingly popular pets. Typically, owners are unaware of the potential risk of ocular injury from the barbed urticating (vascular reaction of the skin associated with itching) hairs found on the dorsal aspect of a tarantula's abdomen. CASE REPORT: A 22-year-old man experienced a red eye with excruciating pain after handling a tarantula. On examination, slitlamp biomicroscopy revealed approximately 30 to 40 barbed tarantula hairs in the conjunctiva, penetrating all layers of the comea. These foreign bodies were causing pain, conjunctival injection, and an anterior chamber reaction. The patient's condition was diagnosed as ophthalmia nodosa and was effectively treated with topical corticosteroids. RESULTS: Patients who manifest red eye and pain after handling a tarantula should be examined to determine if offending barbed hairs are present in the cornea and conjunctiva. To obtain a correct diagnosis, a detailed case history and careful slit-lamp biomicroscopy must be performed.

Molecular form and subcellular distribution of acid beta-galactosidase in fibroblasts from patients with GM1 gangliosidosis, Morquio B disease and galactosialidosis.

The molecular form and subcellular distribution of acid beta-galactosidase in cultured fibroblasts from patients with beta-galactosidase deficiency (GM1-gangliosidosis, Morquio B disease and galactosialidosis) were studied, using antibodies against three different forms of the human enzyme: a high-molecular-weight multienzymic complex, a recombinant 84-kDa precursor, and a 64-kDa tryptic product of the precursor. The mature enzyme from normal fibroblasts was immunoprecipitated by the anti-complex and anti-64-kDa protein antibodies, but not by the anti-84-kDa precursor one. immunofluorescence staining of normal fibroblasts revealed the granular (lysosomal) distribution with anti-64-kDa protein antibody and the perinuclear reticular distribution with anti-84-kDa precursor antibody, probably representing the Golgi apparatus. Both patterns were demonstrated in Morquio B disease, but the residual enzyme activity was exclusively due to the mature enzyme. In Type 1 galactosialidosis, most of the expressed enzyme was detected as the precursor form with a perinuclear reticular distribution. In type 2 galactosialidosis, more than half of the enzyme activity was due to the mature form with a lysosomal distribution. Fibroblasts from a patient with GM1 gangliosidosis, expressing no beta-galactosidase mRNA, did not react against either anti-64-kDa protein antibody or anti-84-kDa precursor antibody. The combined use of immunoprecipitation and immunostaining was useful for analysing the pathophysiology of the intracellular processing and transport of the mutant beta-galactosidase.

Comparison of carboxymethylcellulose vs. hydroxypropyl methylcellulose as a gonioscopic fluid.

BACKGROUND: Hydroxypropyl methylcellulose (HPMC), a popular gonioscopic solution, can cause discomfort and blurred vision. These side effects have been attributed to the preservative benzalkonium chloride (BAK). Carboxymethylcellulose (CMC), an unpreserved, viscous solution, might well provide adequate patient comfort and cushioning during gonioscopy without producing blur or irritation. METHODS: The effects of CMC and HPMC during gonioscopy were evaluated in 55 human subjects. Corneal staining, comfort, subjective vision, and measured visual acuity (VA) were assessed. RESULTS: In comparison with HPMC, CMC effected less corneal staining, greater comfort, and better vision after the procedure, CONCLUSION: CMC proved to be a viable and superior alternative to HPMC as a gonioscopic solution.

Molecular cloning and expression of cDNA for murine galactocerebrosidase and mutation analysis of the twitcher mouse, a model of Krabbe's disease.

The cDNA for a murine galactocerebrosidase was isolated from a murine testis cDNA library on the basis of its homology with the cDNA for human galactocerebrosidase and a PCR method was used to clone the 5' end. It has a 2,278-nucleotide sequence including a 2,004-nucleotide open reading frame, which encodes 668 amino acid residues. The identity between the human and murine amino acid sequences was very high, being calculated to be 84%. Sequencing of cDNA from liver of the twitcher mouse revealed a nonsense mutation at codon 339 (TGG-->TGA). The most abundant mRNA of the murine galactocerebrosidase gave a 3.6-kb band, which was not detected in twitcher mice. This suggests that the cDNA (2,278 bp) we characterized represents a minor species generated by an alternate poly(A) signal and that most of the mRNA has a much longer 3'-untranslated region. Genome analysis revealed that this mutation was homozygous in the twitcher and heterozygous in the carrier but was not present in normal mice. The normal mouse cDNA but not the mutant cDNA of the galactocerebrosidase transfected into COS1 cells gave rise to an increase in enzymatic activity. We concluded that this mutation results in the deficiency of galactocerebrosidase in the twitcher mouse.

Iritis. How to recognize and manage a potentially sight-threatening disease.

A red eye is a common complaint, often related to benign conditions. However, a red eye in conjunction with symptoms such as photophobia, pain, and decreased visual acuity is an important clue to a much more serious disorder. Iritis is one of these but is reversible with proper use of mydriatic and cycloplegic agents and corticosteroids. Heightened clinical suspicion is necessary for timely diagnosis. The examiner should be skilled in the use of a slit lamp or should refer the patient to an ophthalmologist for immediate evaluation. Once the diagnosis is made, treatment with topical corticosteroids is imperative. Close monitoring is required because overuse of corticosteroids has ominous side effects.

Ion-Pair Extraction of Metalloporphyrins into Acetonitrile for Determination of Copper(II).

Equilibrium study of ion-pair extraction of a cationic water-soluble porphyrin [5,10,15,20-tetrakis(1-methylpyridinium-4-yl)porphyrin, H(2)tmpyp(4+)] and its metalloporphyrins (MP) into the acetonitrile layer, separated by addition of sodium chloride (4.00 mol dm(-)(3)) to a 1:1 (v/v) acetonitrile-water mixed solvent, was carried out to develop a new and useful method for the determination of a subnanogram amount of copper(II). M denotes Zn(2+), Cu(2+), Co(3+), Fe(3+), and Mn(3+), and P(2)(-) is porphyrinate ion. The extraction and dissociation constants of the ion-pair complexes, defined by K(ex) = [MP(ClO(4))(4)](org)[MP(4+)](aq)(-)(1)[ClO(4)(-)](aq)(-)(4), K(dis,1) = [MP(ClO(4))(3)(+)](org)[ClO(4)(-)](org)[MP(ClO(4))(4)](org)(-)(1), and K(dis,2) = [MP(ClO(4))(2)(2+)](org)[ClO(4)(-)](org)[MP(ClO(4))(3)(+)](org)(-)(1), were determined by taking into account the partition constant of sodium perchlorate (K(D) = 1.82 ± 0.01). The equilibrium constants were found to be K(ex)K(dis,1) = (7.2 ± 1.3) × 10(4), (6.4 ± 0.9) × 10(4), (1.35 ± 0.13) × 10(5), (4.8 ± 0.6) × 10(3), (1.23 ± 0.05) × 10(4), and (1.42 ± 0.07) × 10(3) at 25 °C for the free base porphyrin (H(2)tmpyp(4+)) and the metalloporphyrins of zinc(II), copper(II), cobalt(III), iron(III), and manganese(III), respectively. The K(dis,2) values were (2.9 ± 1.4) × 10(-)(2), (3.1 ± 1.1) × 10(-)(2), (8.0 ± 4.9) × 10(-)(3), and (5.1 ± 2.2) × 10(-)(2) for the free base porphyrins and the metalloporphyrins of zinc(II), copper(II), and cobalt(III), respectively. The results were developed for determination of a trace amount of copper(II) (3 × 10(-)(8)-4 × 10(-)(6) mol dm(-)(3)) in natural water samples using H(2)tmpyp(4+) with a molar absorptivity of 3.1 × 10(5) mol(-)(1) dm(3) cm(-)(1) at a precision of 1.3% (RSD). The determination of copper(II) was not interfered by the presence of 10(-)(4) mol dm(-)(3) of Mn(2+), Co(2+), Ni(2+), Hg(2+), Cd(2+), Ag(+), Cr(3+), V(5+), Al(3+), Mg(2+), Ca(2+), Br(-), I(-), SCN(-), and S(2)O(3)(2)(-) and 10(-)(5) mol dm(-)(3) of Fe(3+), Zn(2+), and Pd(2+).

Molecular defects in Krabbe disease.

Krabbe disease (globoid cell leukodystrophy) is an autosomal recessive neurodegenerative disorder that affects both the central and peripheral nervous systems due to an enzymatic defect of the galactocerebrosidase. In this study, molecular defects in Krabbe disease were investigated in 11 patients (seven Japanese and four non-Japanese) using cultured skin fibroblasts. A Japanese late infantile patient had a missense mutation of Pro at codon 302 to Ala and a non-Japanese patient had a missense mutation of Val at codon 550 to Gly. The reduced enzymatic activities expressed from the cDNAs with these missense mutations and from the previously reported nonsense mutation (E369X, Glu at codon 369 to stop codon) were confirmed. Genomic DNA analyses revealed that the P302A and E369X mutations were heterozygous and the V550G mutation was homozygous in these patients. A 12 base deletion with a 3 base insertion was found in three unrelated Japanese infantile patients, but not in 30 controls. The mutation was homozygous in two patients and heterozygous in one patient. We could not find any confirmed mutation in the coding region in the other six patients. These findings suggest that mutations in infantile and late infantile patients are relatively heterogeneous.

Update on cholesterol and the eye.

High serum cholesterol levels have been accepted as a major risk factor for cardiovascular disease and arteriosclerosis. There are several corneal signs that potentially indicate abnormal cholesterol levels, thus altering the eye care practitioner to refer the patient for lipid evaluation. This article describes the corneal manifestations of abnormal cholesterol levels and offers guidelines for management.

Krabbe disease: isolation and characterization of a full-length cDNA for human galactocerebrosidase.

Human galactocerebrosidase, the enzyme deficient in Krabbe disease, was purified, through several hydrophobic column steps and gel filtration, 22,650-fold from human lymphocytes. Using information on its N-terminal and internal amino acid sequences, and the polymerase chain reaction method, we cloned a full-length cDNA for the enzyme. The deduced amino acid sequence matched all amino acid sequences determined. The 3780 nucleotide sequence included 2007 nucleotides which encoded a single chain peptide of 669 amino acid residues with a 26 amino acid N-terminal signal peptide and six potential asparagine-linked glycosylation sites. The galactocerebrosidase cDNA detected an about 4 kb mRNA band material in human cultured skin fibroblasts. A nonsense mutation was found at codon 369 (GAA-->TAA) in the coding sequence of cDNA amplified from cultured skin fibroblast mRNA from a patient with typical Krabbe disease.

Effects of double amino-acid substitution polymorphism in acid beta-galactosidase gene in two inbred strains of mice.

We observed earlier that there are 5 nucleotide polymorphisms in the protein coding sequence of the acid beta-galactosidase gene between the C57BL/6J and DBA/2J strains of mice. Two of them result in amino acid substitutions. Consequences of the difference in the primary amino acid sequence were studied by introducing the two DBA polymorphisms into the C57BL cDNA, individually and in combination, by oligonucleotide-directed mutagenesis and expressing the resultant cDNAs in the COS-1 cell expression system. Introduction of one polymorphism, Asn517-->Asp into the C57BL cDNA, did not alter the acid beta-galactosidase activity in the transfected COS-1 cells, while introduction of Gly539-->Arg completely abolished the catalytic activity. When both polymorphisms were introduced together, as in the DBA mice, however, the acid beta-galactosidase activity was restored to that of the C57BL level. Thus, Asn517-->Asp appears to counteract the activity-abolishing effect of Gly539-->Arg, although it does not by itself raise the catalytic activity. All four types of cDNA generated similarly large amounts of stable mRNA in COS-1 cells. These results do not explain the significantly low acid beta-galactosidase activity in tissues of DBA mice, described earlier and also confirmed in this study.

A case of Hallervorden-Spatz disease: progressive and intractable dystonia controlled by bilateral thalamotomy.

We present a 10-year-old girl with Hallervorden-Spatz disease diagnosed clinically from the neurological manifestations and the characteristic MRI findings. Her main symptom, dystonia, was progressive and resistant to medication, but this dystonia was controlled by bilateral thalamotomy. No clinical progression of the symptoms was recognized at 21 months from the last operation.

A case of pigmentary type of orthochromatic leukodystrophy with early onset and globoid cells.

We report herein a sporadic case of the pigmentary type of orthochromatic leukodystrophy with early onset and very rapid clinical course. The patient's development was normal until 2 years old, when he experienced visual disturbance. Rapid deterioration resulted in death 1.5 years after the onset. Metachromatic leukodystrophy, globoid cell leukodystrophy and adrenoleukodystrophy were excluded by biochemical assays. Autopsy findings were compatible with the diagnosis of the pigmentary type of orthochromatic leukodystrophy. However, there were unique findings of severe neuronal loss and the collection of globoid-like cells in the interface of the gray matter and the white matter. Immunohistochemical staining of myelin basic protein, proteolipid protein and galactocerebroside demonstrated that these myelin constituents were equally preserved in the posterior column, while absent in the lateral and anterior columns of the spinal cord.

GM1-gangliosidosis (genetic beta-galactosidase deficiency): identification of four mutations in different clinical phenotypes among Japanese patients.

GM1-gangliosidosis is a genetic neurological disorder caused by mutations in the lysosomal acid beta-galactosidase gene. While its phenotypic expression is complex, it is usually classified as being of infantile, juvenile, or adult form, on the basis of age at onset, the rate of symptomatic progression, and severity of central nervous system involvement. We have analyzed the acid beta-galactosidase gene in 12 Japanese patients from nine families. The aim was to identify mutations in individual patients and then to examine possible correlation between the mutations and the clinical phenotypes. Northern blotting studies with a full-length human beta-galactosidase cDNA showed that the mRNA ranged from undetectable to substantially decreased in the infantile patients but was normal in quantity and size in all juvenile and adult patients. Four distinct missense mutations have been identified, each limited to the respective clinical forms within our small-size samples. In the infantile patient with decreased but detectable mRNA, a point mutation was found resulting in Arg49----Cys. In the infantile patient with nearly undetectable mRNA, mutation Arg457----Ter was identified. The mutation Arg201----Cys was found in all four of the juvenile patients, while all six adult patients were homozygous for the point mutation Ile51----Thr. The mutations found in the juvenile and adult patients alter restriction sites in the normal gene and thus are amendable to quick screening. The prediction that these mutations are responsible for the clinical disease was confirmed by no expression of the catalytic activity of the mutant proteins in the COS-I cell expression system.(ABSTRACT TRUNCATED AT 250 WORDS)

Expression of the beta-hexosaminidase alpha subunit gene with the four-base insertion of infantile Jewish Tay-Sachs disease.

One of the two mutations responsible for the classical infantile Jewish form of Tay-Sachs disease is a four-base insertion in exon 11 of the beta-hexosaminidase alpha subunit gene. The gene is known to be transcribed normally, but the mRNA is essentially undetectable. It is not clear why such a relatively minor abnormality results in complete failure to generate stable mRNA. The four-base insertion was introduced into the normal beta-hexosaminidase cDNA by site-directed mutagenesis. When COS-1 cells were transfected with the resultant mutant cDNA, it generated stable mRNA and a truncated, relatively stable but catalytically inactive enzyme protein. The mutant enzyme protein was not processed nor released into the culture medium. The mutant cDNA also generated the truncated enzyme protein in an in vitro translation system with rabbit reticulocyte lysate. COS-1 cells transfected with a 3' end of the gene segment, from intron 8 through the 3' terminus, generated processed RNA of approximately 2 kilobases, the size expected from normal splicing, irrespective of presence or absence of the four-base insertion in exon 11. These results indicate that the four-base insertion does not destabilize properly spliced mRNA, nor does it interfere with normal splicing of the transcript, at least in the expression system utilized. If the four-base insertion is responsible for the undetectable mRNA in the mutant cells, it must interfere with some other steps in the processing/splicing/transport of the primary transcript yet to be examined. On the other hand, the possibility cannot be excluded definitively that another still unidentified abnormality in the same allele might be responsible for the nearly complete absence of mRNA.

Genetic cause of a juvenile form of Tay-Sachs disease in a Lebanese child.

Abnormality in the beta-hexosaminidase alpha gene underlying the clinical phenotype of a Lebanese patient with a juvenile form of Tay-Sachs disease has been studied. Clinical features were progressive spasticity, ataxia, and cognitive decline. The protein coding sequence of several beta-hexosaminidase alpha-chain complementary DNAs isolated by polymerase chain reaction was completely normal except for a G-to-A transition at nucleotide position 1511 within exon 13, which resulted in substitution of the normal arginine 504 (CGC) with histidine (CAC). Although the patient was from a first-cousin marriage, she was heterozygous for this mutation. The abnormality in the other allele, which is carried by the father, was not identified, except that it is neither of the two mutations responsible for the infantile Jewish Tay-Sachs disease. Biosynthetic and immunoprecipitation studies in cultured fibroblasts showed synthesis of the alpha-chain precursor, but the mature form of the alpha-subunit was not detected.

Age-related response of male melon fliesDacus cucurbitae (Diptera: Tephritidae) to cue-lure.

Laboratory-reared and wild adults of the melon fly,Dacus cucurbitae Coquillett, were tested for response to cue-lure at various ages. Virgin laboratory (4, 6, 8, 10, 12, and 14 days old) and wild (10, 12, 14, 16, 18, 20, and 22 days old) flies were released into outdoor field cages and trapped from 0800 until 1600 hr. Response of males to cue-lure increased with age and corresponded with sexual maturity for each strain. Females of both strains were relatively nonresponsive to cue-lure. Failure to eradicate in past male annihilation programs againstD. cucurbitae may be explained in part by the fact that only older males, which may have already mated with gravid females, respond to cue-lure.