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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

[Scanning electron microscopic observation of hydrodissected human lens nucleus].

Using a scanning electron microscope, we studied the surface structure of 5 different central nuclei, isolated by repeated hydrodissection during extracapsular cataract extraction. Aside from the damage induced by surgical maneuvers, the surfaces were very smooth, and there was no major disruption of the lamellar structure of the lens. Our studies have shown that hydrodissection, which allows atraumatic separation of the lens fiber layers, can be used to analyze the lamellar structure of the lens. Some of the evaluated specimens showed a distinct suture line and the lens fibers were approximately at right angles to it. These observations were different from those reported previously.

[Histopathologic study of after-cataract in the pseudophakic rabbit eye using out-of-the-bag fixation. (I)].

We performed light microscopic studies of after-cataract formation after phacoemulsification and out-of-the-bag IOL implantation in 23 rabbit eyes. One day after, epithelial cells at the equatorial area began to move to the pupillary area of the posterior capsule. A week later, we saw adhesion of the anterior capsular edge to the central posterior capsule with newly-formed lens fibers in the capsular space. These findings were similar to after-cataract formation after phacoemulsification only. We found no apparent effect of IOL on after-cataract formation when it was fixed out of the bag.

[Histopathologic study of after-cataract in the pseudophakic rabbit eye using in-the-bag fixation. (II)].

Sequential changes in the lens capsules of the rabbit eye after phacoemulsification with in-the-bag fixation of IOL were studied light microscopically. There were three types of after-cataract. Two of them, in which the anterior capsular flap contacted the posterior capsule and/or the anterior surface of the optics of IOL, were similar to that in aphakic cases. The other type, in which the anterior capsular edge contacted the IOL only, showed a number of characteristic findings. The capsular space was closed with a residual lens capsule and IOL optics, and typical Soemmerring's ring was observed there. Fibrous proliferation was observed at the anterior capsular edge, not on the posterior capsule. Lens epithelia on the posterior capsule moved beneath the IOL and reproduced new lens fibers.

[Pathology of IOLs placed in the capsular bag of white rabbit eyes].

The authors performed a histopathological study to observe the surface of IOLs placed inside the capsular bag of white rabbit eyes. While a cellular reaction to the haptics was indicated by lens epithelial cells and collagen fibers surrounding the haptics, a wound healing reaction at the anterior capsule-optic junctions was suggested by an extensive amount of spindle cells and collagen fibers. However, collagen fibers and degenerated cells on the optics could be seen only under electron microscopy. Regenerated lens fibers, lens epithelial cells, and proliferation of spindle cells were noted between the posterior side of the optics and the posterior capsules.

[Anatomical evaluation of hydrodissected human lens nucleus].

We measured the diameter and central thickness of the central nucleus, isolated by repeated hydrodissection, in 100 eyes undergoing extracapsular cataract extraction. The average diameter and central thickness were 6.43 +/- 0.86 mm and 2.93 +/- 0.36 mm, respectively. The average ratio between the two measurements was 2.12, which is similar to that of an extracted crystalline lens. Statistical analysis revealed a positive correlation between central thickness and age (p < 0.05). There was a weak positive correlation between central thickness and hardness of the nucleus. Since specimens were obtained from clinical cases in this study, further evaluation should be made using cadaver eyes.

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.