Lecithin-cholesterol acyltransferase (LCAT) deficiency without mutations in the coding sequence: a case report and literature review.

Familial lecithin-cholesterol acyltransferase (LCAT) deficiency (FLD) is a rare genetic disease characterized by corneal opacities, normocytic anemia, dyslipidemia, and proteinuria progressing to chronic renal failure. In all FLD cases, a mutation has been found in the coding sequence of the LCAT gene. FLD is clinically distinguished from an acquired form of LCAT deficiency by the presence of corneal opacities. Here we describe a 36-year-old woman presenting with clinical, pathological, and laboratory data compatible with FLD. Her mother and elder sister had corneal opacities. However, genetic analysis revealed there were no mutations in the LCAT coding sequences and no alterations in LCAT mRNA expression. Furthermore, we were unable to find any underlying conditions that may lead to LCAT deficiency. The present case therefore demonstrates that LCAT deficiency may be caused by factors other than mutations in the coding sequence and we suggest that a translational or posttranslational mechanism may be involved.

Visual results and complications of temporal incision phacoemulsification performed with the non-dominant left hand by junior ophthalmologists.

AIMS: To assess the results of temporal incision phacoemulsification and aspiration performed with dominant and non-dominant hand of ophthalmology trainees. METHODS: Retrospective analysis were made of 203 surgeries with dominant hand and 207 with non-dominant by five trainees at two institutions. Trainees sat at the patient's head, manipulating instruments with the dominant right hand for the right eye, and the non-dominant left hand for the left eye. RESULTS: Vitreous loss occurred in 12 (5.9%) of 203 dominant operated eyes and seven (3.4%) of 207 non-dominant operated eyes. The rate of endothelial cell loss was 6.1% (9.8%) in dominant and 7.4% (12.4%) in non-dominant. Mean ultrasound time were 1.81 (0.70) minutes in dominant and 1.78 (0.78) minutes in non-dominant. One trainee showed statistically significant excesses in incidence of vitreous loss in dominant operated eyes (8.7%, p=0.0270), and one showed statistically significant prolongation of the operation in nondominant operated eyes (26.3 minutes, p=0.0315). In all other trainees, all parameters had no difference in both sides. CONCLUSIONS: Ophthalmology trainees could successfully learn the technique with both hands. The authors consider that the skill of the non-dominant hand may be knowledge based and that surgeons avoid mistakes by mental efforts.

Reduced expression of focal adhesion kinase in liver metastases compared with matched primary human colorectal adenocarcinomas.

The focal adhesion kinase (FAK) is implicated in integrin-mediated signal transduction pathways used in cell adhesion, cell motility, apoptosis, and anchorage-independent growth. Because cancer invasion and metastasis are thought to be associated with alterations in cellular adhesive and motile properties, we studied the expression of four focal adhesion proteins including FAK in matched samples of human normal colorectal mucosa (N), primary colorectal adenocarcinomas (T) and liver metastases (M) from 10 patients by Western blot analysis. This gave us the advantage of directly comparing levels of focal adhesion protein expression within the same genetic background. Average FAK expression level was significantly higher in T than in N and it was significantly lower in M than in T. Average paxillin expression level was also significantly higher in T than in N, but it was not significantly different between T and M. Similar results were obtained by immunohistochemical analyses of FAK and paxillin expression. Average vinculin and talin expression levels showed no significant differences among these three samples (N, T, and M). These data demonstrate that the FAK expression level increases in primary tumors compared with normal mucosa and decreases in liver metastases to the level of normal mucosa in the majority of human colorectal adenocarcinomas. Up- and down-regulation of FAK protein expression observed in this study may have a profound effect on the signal transduction.

Results of vitrectomy performed at the time of phacoemulsification complicated by intravitreal lens fragments.

AIM: To evaluate outcome of vitrectomy performed at the time of phacoemulsification complicated by intravitreal lens material. METHODS: Clinical records associated with consecutive 8536 phacoemulsification procedures were reviewed retrospectively. RESULTS: 17 (0.20%) eyes had a posterior capsule rupture with retained lens material in the vitreous cavity that required vitrectomy. Final visual acuity was 0.5 or better in 14 eyes (82%) and 0.4 to 0.1 in three eyes (18%). Retinal detachment occurred in one eye during vitrectomy and two after the surgery. Cystoid macular oedema was observed in two eyes and none developed glaucoma. The corneal endothelial cell loss was 5.7% (SD 6.8 %) (n=15) at 3-6 months postoperatively. CONCLUSIONS: Combined vitrectomy and intraocular lens implantation at the time of phacoemulsification complicated by intravitreal lens material is an option to be considered to reduce the risk of postoperative complications including secondary glaucoma and corneal endothelial cell damage.

Y-27632, an inhibitor of rho-associated protein kinase, suppresses tumor cell invasion via regulation of focal adhesion and focal adhesion kinase.

Migration of rat ascites hepatoma (MM1) cells, invasion and phagokinetic movement were induced by the combination of lysophosphatidic acid (LPA) and fibronectin (FN). Induction of migratory activity was tightly correlated with morphological change of MM1 cells from spherical or polygonal-shaped cells to fusiform-shaped ones with pseudopodia. MM1 cells were mobile in a fusiform shape, whereas those of a spherical or polygonal shape were not. A small GTPase Rho and one of its downstream effectors ROCK (Rho-associated coiled-coil forming protein kinase), play essential roles in these processes, as evidenced by suppression of migration and morphological change of MM1 cells by Clostridium botulinum C3 exoenzyme, an inhibitor of Rho, or by Y-27632, an inhibitor of ROCK. Y-27632 also suppressed the formation of fusiform-shaped pseudopodia-carrying MM1 cells that was induced by stimulation with the combination of LPA and FN. LPA and FN also evoked the formation of focal adhesions and actin bundles, and tyrosine phosphorylation of focal adhesion kinase (FAK) and paxillin. The inhibitory effect of Y-27632 on LPA-induced migration and morphological change of MM1 cells was considered to be mediated, at least in part, by impaired formation of focal adhesions and actin bundles. Y-27632 suppressed LPA-induced tyrosine phosphorylation of FAK and paxillin, suggesting that ROCK regulates these molecules and Y-27632 inhibits cellular migration and morphological change, at least in part, through this regulation.

Serum levels of soluble IL-2 receptor, IL-12, IL-18, and IFN-gamma in patients with acute graft-versus-host disease after allogeneic bone marrow transplantation.

BACKGROUND: Acute graft-versus-host disease still represents the major factor that limits successful allogeneic bone marrow transplantation. Cytokines released by type 1 T-helper cells are thought to play a pivotal role in acute graft-versus-host disease. OBJECTIVE: This study was performed to investigate whether the serum levels of soluble IL-2 receptor, IL-12, IL-18, and IFN-gamma were associated with the manifestation of acute graft-versus-host disease. METHODS: Serum cytokine levels were measured by sandwich ELISA in 18 patients who underwent allogeneic bone marrow transplantation. RESULTS: Serum levels of soluble IL-2 receptor, IL-12, IL-18, and IFN-gamma were increased in patients in whom acute graft-versus-host disease developed. However, only serum soluble IL-2 receptor levels were significantly related to disease severity. Serum levels of IL-12 and IL-18, both of which are mainly produced by activated macrophages, were increased in different phases of acute graft-versus-host disease, especially grade I. Serum levels of soluble IL-2 receptor and IFN-gamma were significantly elevated in patients with fever. CONCLUSION: Serum levels of soluble IL-2 receptor were more closely related to the severity of acute graft-versus-host disease than those of IL-12, IL-18, and IFN-gamma.

Noncontact specular microscopy of human lens epithelium.

PURPOSE: To obtain in vivo specular images of human lens epithelial cells (LECs) from persons with or without age-related cataract (ARC); to identify features that describe individual aspects of these complex images; to develop feature scales to quantify the severity of each feature; and to study the association of these features with LEC count, age, Lens Opacity Classification System III (LOCS III) classifications and microscopic features of lens epithelium in ARC. METHODS: One hundred fifty-two individuals underwent ophthalmic examinations and LOCS III cataract classifications. Specular images of lenses were captured using a modified noncontact corneal specular microscope (SML-2; Konan, Hyogo, Japan). Enhanced images were graded in a masked fashion, and the presence or absence and severity of each of four features in the specular image ("columnar organization," "linear furrows," "puffy clouds," and "black holes") was graded on a four-step scale. The generalized linear model with intraclass correlation was used to ascertain the statistical significance of associations between age, sex, LOCS III grade, cell count, and feature grade. Capsulorrhexis specimens from 29 patients were studied with correlative light and electron microscopy. RESULTS: LEC density declined with age and was inversely correlated with the scalar grade for puffy clouds and for the size and number of black holes. The scalar grade for columnar organization was inversely associated with the severity of posterior subcapsular and nuclear cataracts, which was the only feature associated with the LOCS III grade of ARC. No statistically significant associations were found between average cell count and LOCS III grade. CONCLUSIONS: With the use of the corneal specular microscope excellent in vivo specular images of the LECs were obtained, the features in these images that correlated well with microscopic findings were classified, and cell density in vivo was estimated.

Cooperation of fibronectin with lysophosphatidic acid induces motility and transcellular migration of rat ascites hepatoma cells.

We have previously shown that the transcellular migration of rat ascites hepatoma (AH130-MM1) cells through a cultured mesothelial cell monolayer (MCL) is triggered with lysophosphatidic acid (LPA) that stimulates actin polymerization and myosin light chain phosphorylation through the activation of Rho-ROCK (Rho-kinase) cascade. When, however, the motility of MM1 cells on a glass surface was tested by phagokinetic track motility assay, LPA failed to induce the motility. Nevertheless, when the glass had been coated with fibronectin (FN), LPA could induce phagokinetic motility which was accompanied by transformation of MM1 cells to fusiform-shape and assembly of focal adhesion. beta1 integrin, the counter receptor of FN, was expressed on MM1 cells. Anti-FN antibody, anti-beta1 integrin antibody and cyclo-GRGDSPA remarkably suppressed LPA-induced phagokinetic motility. These antibodies suppressed LPA-induced transcellular migration through MCL, as well. These results indicate that actin polymerization and phosphorylation of myosin light chain through Rho activation are insufficient for inducing motility but the cooperative FN/beta1 integrin-mediated adhesion is necessary for both the phagokinetic motility and transcellular migration of MM1 cells.

Involvement of small GTPases Rho and Rac in the invasion of rat ascites hepatoma cells.

Lysophosphatidic acid (LPA) triggers the invasion of a mesothelial cell monolayer by rat ascites hepatoma (MM1) cells. LPA also induces rapid morphological changes of MM1 cells, cell surface blebbing and pseudopodia formation. Pseudopodia formation is tightly correlated with cellular invasiveness. Clostridium Botulinum C3 exoenzyme and genistein abrogated the formation of blebs and pseudopodia together with the inhibition of invasion, indicating that GTPase Rho and certain tyrosine kinases are involved in both processes. MM1 cells expressing constitutively active Rho exhibited the invasion and the formation of blebs and pseudopodia in the absence of LPA. In contrast, MM1 cells expressing constitutively active Rac were not invasive in the absence of LPA, but were invasive in the presence of LPA. Their morphological response to LPA was almost the same as that of parental MM1 cells. Expression of dominant negative Rac suppressed the invasiveness to approximately 3% of that of parental MM1 cells, together with the inhibition of pseudopodia formation. Thus, Rho and Rac are cooperatively involved in both the invasion and the related morphological changes of MM1 cells. Rho activation is sufficient both for the induction of invasion and the morphological changes leading to the invasion, whereas Rac activation is necessary but not sufficient by itself. We propose that Rho activation is not mediated by Rac but the cooperation of both GTPases is essential to trigger the invasive behavior of MM1 cells.

Inhibition of tumor invasion and metastasis by a novel lysophosphatidic acid (cyclic LPA).

Fetal calf serum (FCS) and 1-oleoyl lysophosphatidic acid (LPA) were previously found to be potent inducers of invasion (transcellular migration) in an in vitro system. A novel LPA, composed of cyclic phosphate and cyclopropane-containing hexadecanoic acid (PHYLPA), first isolated from myxoamoebae of Physarum polycephalum, and its synthetic derivatives (cLPA) were tested for their ability to inhibit tumor cell invasion and metastasis. Amoung these, Pal-cLPA, which has a palmitoyl moiety, was most potent in inhibiting invasion, with 93.8% inhibition at the concentration of 25 microM. Invasion in vitro by mouse melanoma cells (B16), human pancreatic adenocarcinoma cells (PSN-1), human lung cancer cells (OC-10) and human fibrosarcoma cells (HT-1080) was also inhibited by Pal-cLPA. The stimulation of MMI cells with LPA triggered F-actin formation, which was impaired by the addition of Pal-cLPA at invasion-inhibitory concentration. Pal-cLPA induced a rapid increase in adenosine 3',5'-cyclic monophosphate (cAMP) concentration in MMI cells. The addition of dibutyryl cAMP significantly abrogated LPA-induced invasion by MM1 cells and actin polymerization in the cells. The inhibition of MM1 cell invasion by Pal-cLPA may be ascribed to an increased level of cAMP. Pal-cLPA also suppressed invasion in vitro by MM1 cells induced by FCS dose dependently, without affecting proliferation. It also suppressed the pulmonary metastasis of B16 mouse melanoma cells injected into the tail vein of C57BL/6 mice. Thus, Pal-cLPA is effective in inhibiting invasion and metastasis of a variety of tumor cells.

Characterization of a 34 kDa immunoreactive peptide with the anti protein kinase C antibody which can recognize part of the C4 region.

Among the immunoreactive peptides from hamster tissues with the antibody (Ab-1) which can recognize part of the C4 conserved region of protein kinase C (PKC), a peptide with 34 kDa (34 kDa species) is constitutively detected in the soluble and the nuclear but not in the particulate fractions. The partially purified 34 kDa species demonstrates no phospholipid-dependent or independent PKC activity and no phorbol 12,13 dibutyrate (PDBu) binding, and is not phosphorylated under the assay condition of PKC activity. The species inhibits the enzymic activity of the partially purified PKC from hamster brain in an uncompetitive manner with Ki of 200 +/- 23 nM.

Molecular cloning and sequence analysis of the cDNA for the mouse counterpart to adult hamster liver purified growth inhibitory factor.

A cDNA clone encoding the mouse counterpart to adult hamster liver purified growth inhibitory factor (PGIF) was isolated from a mouse liver cDNA library by using antibodies raised against PGIF and sequenced. It contained a single open reading frame with a coding capacity for a 323 amino acid protein. Sequence analysis showed that it shared high homology with rat- and human liver arginases: the cDNA clone was 92% identical for rat arginase at the nucleotide level and was 93% identical to it at the deduced amino acid level. These results suggest that PGIF derived from adult hamster liver was identical or closely related to an isoform of hamster liver arginases.

Distribution and subcellular localization of a growth inhibitory factor in hamster liver and its intracellular partner(s).

The subcellular, intralobular distributions and intracellular partner(s) of a factor which inhibits the proliferation of cell growth (Hashimoto C. et al. (1994) Biochim. Biophys. Acta 1221, 107-117) were determined in hamster livers, using a combination of immunological and biochemical techniques. The IgG fraction from an antiserum raised against the growth inhibitory factor with 37 kDa was shown to be highly specific for the antigen. The nuclear and cytosolic fractions demonstrated inhibitory effects on cell growth and Western blot analysis revealed that both fractions contained the immunoreactive 37 kDa protein with the anti-inhibitory factor IgG but microsomal and mitochondrial fractions did not. The nuclear and cytoplasmic localization of the inhibitory factor were further confirmed by immunochemical staining mediated through the immune IgG and an avidin-biotinylated horseradish peroxidase complex, the parenchymal liver cells were clearly stained, but endothelial and connective tissue cells were not. Although some staining was evident throughout the liver parenchyma, the hepatocytes with most intensively stained nuclei were located in the periportal region. In the liver from hamsters 6 days old or the regenerating hamster livers 3 days after partial hepatectomy, the staining intensity was low and the number of hepatocytes with the inhibitory factor positive nuclei was very few compared with the adult hamster livers. In primary cultures of the isolated hepatocytes from adult hamster the inhibitory factor disappeared from nuclei after incubation for 24-48 h. The extracts of hepatic nuclei from adult hamsters were immunoprecipitated with either the anti-growth inhibitory factor IgG or a monoclonal antibody to the RM protein. The growth inhibitory factor and the RB protein coprecipitated in each case, implying that the proteins were complexed with each other in the nuclei. The RB protein family is composed of two sets of species, an un- or underphosphorylated species and a hyperphosphorylated one. It was suggested that the factor bound preferentially to the un- or underphosphorylated member of the family.

Postpartum central retinal artery occlusion simulating Purtscher's retinopathy.

A 30-year-old woman noticed sudden visual loss in her right eye 3 days after a normal childbirth and without eclampsia during pregnancy. An ophthalmic examination revealed that she had impending central retinal artery occlusion and her right visual acuity was 20/2000, which recovered to 20/30. A systemic examination showed hypercoagulability and hyperlipidemia but no abnormal findings on brain CT scan and echocardiography. Clinical features of this case were very similar to Purtscher's retinopathy.

Anterior capsulectomy using an ordinary phacoemulsification tip (phaco-capsulectomy).

I performed an anterior capsulectomy in six eyes using an ordinary phacoemulsification (phaco) tip. The technique, which involves inserting a beveled-down phaco tip directly into the lens, created smooth and tear-free capsular openings. Although the procedure needs to be refined, I believe a phaco tip can be used effectively and easily to remove the anterior capsule.

Isolation and characterization of a growth inhibitory factor from hamster liver.

Among lysates from various organs and tissues of adult hamsters only lysates from liver demonstrated an inhibitory effect on the cell growth of SV40-transformed hamster fibroblasts in culture. Lysates from the liver of fetal hamsters and those from 7-day-old hamsters did not demonstrate any inhibitory effect on the cell growth. Lysates from the remnant liver 3 days after partial hepatectomy did not show any inhibitory effect on the cell growth but lysates from the remnant liver 14 days after the operation came to show an appreciable inhibitory effect on the cell growth. An inhibitor of the cell growth was purified from adult hamster liver by ammonium sulfate precipitation, DEAE-, hydroxyl apatite-, phenyl Sepharose- and Sephadex G75 column chromatography. The cell growth inhibitor thus prepared was shown to be pure by an ion-exchange chromatography, SDS-PAGE and analytical isoelectric focusing. The inhibitor was found to have a molecular mass of 37 kDa and an isoelectric point of approx. 7.5 and to cause reversible arrest of the transformed fibroblasts predominantly in the G0/G1 phase of the cell cycle at the concentration of approx. 0.9 microgram per ml.