Non-coherent light for photodynamic therapy of superficial tumours in animals.

Cultured 9L cells were incubated with varying concentrations of pheophorbide-a-hexyl ether (HPPH) and then exposed to 665-nm red light from a non-coherent light source or a dye laser. Cell death was produced by both light sources, with the non-coherent light being most effective at the highest HPPH concentrations. To assess the feasibility of using the non-coherent light source for clinical photodynamic therapy (PDT), four dogs and three cats presenting with spontaneous superficial tumours were injected intravenously with 0.15 mg kg(-1) of HPPH, 1 h before their tumours were irradiated with 665-nm non-coherent light (50 mW cm(-2), 100 J cm(-2)). Of the nine tumours treated, there were eight complete responses, all occurring in animals with squamous cell carcinoma. After 68 weeks of follow-up, the median initial disease-free interval had not been reached. These data suggest that non-coherent light sources may be efficacious for photodynamic therapy of spontaneous superficial tumours in animals, representing a cost-effective alternative to medical lasers in both veterinary and human oncology.

Preclinical evaluation of 5-aminolevulinic acid-based photodynamic therapy for canine transitional cell carcinoma.

As a prelude to photodynamic therapy, 5-aminolevulinic acid (ALA) was given orally to healthy dogs. ALA-induced protoporphyrin IX (PpIX) fluorescence significantly increased in the mucosa of the urinary bladder in an ALA dose-dependent fashion. Vomiting occurred after ALA administration in 70% of the dogs but did not affect PpIX fluorescence. ALA-based photodynamic therapy (PDT) of the urinary bladder in healthy dogs caused only submucosal oedema within the bladder wall. No haematologic or serum biochemistry abnormalities were observed after ALA administration. Microscopic haematuria was observed in all the dogs after PDT but was mild and self limiting. ALA-based PDT was administered to six dogs with transitional cell carcinoma (TCC) of the lower urinary tract. ALA-based PDT resulted in tumour progression-free intervals from 4 to 34 weeks in five dogs; one dog with pre-existing hydronephrosis died shortly after PDT. Dogs with TCC represent an outbred, spontaneous, tumour model for developing PDT protocols for humans with bladder cancer.

Laser lithotripsy of a urethral calculus via ischial urethrotomy in a steer.

A steer examined because of obstructive urolithiasis and urethral rupture underwent laser lithotripsy, using a chromium-thulium-holmium:yttrium-aluminum-garnet (Ho:YAG) laser inserted through an ischial urethrotomy. Procedures were performed with caudal epidural anesthesia. Six months after surgery, the urethra was patent with no clinical evidence of urethral stricture or fistula. Ischial urethrotomy provided rapid access to the bladder for catheterization and to the obstructive urolith for lithotripsy. Laser lithotripsy was a rapid and effective means of urolith removal in this steer.

Functional opening of the fourth ventricular outlet in C57BL/6J and delayed splotch mouse embryos.

To compare the functional development of the fourth ventricular outlet in the myeloschisis-Chiari malformation complex with that of a normal brain, the chronological development of the outlet in C57BL/6J non-neural-tube defect mouse embryos was examined as the first step. Then we compared the results with those of homozygotic delayed splotch (Spd) mouse embryos which had neural-tube defects (NTDs). Ferrous chloride (Prussian blue) solution was injected into the lateral or mesencephalic ventricle on gestation days 13-16 in the case of control C57BL/6J mouse embryos and on gestation days 14-16 in the case of homozygotic Spd mouse embryos which had open spinal NTDs and hindbrain anomalies comparable to human Chiari malformation. At 30 min after the injection, acid fixative was infused through the heart to set off the Prussian blue reaction, which makes the dye visible by the precipitation of ferric chloride. According to the present method, more than 75% of C57BL/6J mouse (non-NTD control) embryos showed the evidence of function of the fourth ventricular (4V) outlet from gestation day 15. It was difficult to apply the same method to Spd mouse embryos with NTDs due to the small size of ventricles. Only 4 injections were successful, of which 3 showed the functioning evidence of the 4V outlet. Though the number of mouse embryos with NTDs studied was small, the results suggest that the chronological progress of functional opening of the fourth ventricle in mouse embryos with NTDs is similar to that of control non-NTD embryos.

Concanavalin-A-induced open neural tube defects in chick embryos.

Open neural tube defects developed in 12 of 122 alive chick embryos treated with exogenous lectin (concanavalin-A) at stages between 10 or 14 as defined by Hamburger and Hamilton. Embryos treated at stage 10, the time of anterior neuropore closure, developed exencephaly or extensive neural openings from the level of rhombencephalon to the thoracic spinal cord, while embryos treated at stages between 11 and 14, at posterior neuropore closure, developed only small myeloschisis in the thoracolumbar region. The failure of neural tube closure at a critical time is a major cause of neural tube defects.

The use of ethanol for preserving proteins and glycoproteins of the trabecular meshwork.

We evaluated methods for storage of the trabecular meshwork and irisciliary body for protein and glycoprotein analysis. The trabecular meshwork and irisciliary body of rabbit eyes were microdissected and stored in Laemmli sample buffer, Carnoy's fluid (75% ethanol-25% glacial acetic acid), or 100% ethanol at ambient temperature, 4 degrees C, -20 degrees C, or -80 degrees C for 24 h or 30 days. Fresh and stored tissues were processed for one-dimensional polyacrylamide gel electrophoresis (PAGE) and Western blot using Con A lectin. The protein patterns of stored and fresh tissues as determined by silver-stained polyacrylamide gels were similar. However, ethanol-stored tissues revealed other proteins (MW of 15-30 kD and 150 kD), and the staining intensity and band resolution of lower MW (15-40 kD) were enhanced. The glycosylation patterns of stored and fresh tissues as determined by Con A (recognizes certain N-linked glycoproteins containing mannose and glucose) were also similar, but the ethanol-stored tissues stained more intensely, especially the high (>200 kD) and low (<35 kD) MW ranges. These PAGE results indicate that ethanol storage is useful for preserving and resolving the protein/glycoprotein profiles of the trabecular meshwork.

Ultrastructural alterations in the aqueous outflow pathway of adult buphthalmic rabbits.

The aqueous outflow pathway of adult rabbit eyes with congenital glaucoma (buphthalmos) was examined by light microscopy and by scanning and transmission electron microscopy. The morphology of the buphthalmic rabbit aqueous outflow pathway was markedly abnormal when examined at 6 months, 1 yr, and 2 yr displaying apparent loss and/or compression of the iris pillars, dilation of the intertrabecular spaces, loss of endothelial cell-to-cell association and disorganization of trabecular lamellae, and posterior displacement of the aqueous plexus. In addition, the trabecular meshwork lamellae were observed only adjacent to the sclera and the inner portion of the trabecular meshwork was limited to swirls of collagen with scattered cells. These morphological findings suggest that the disease process in the rabbit principally involves an alteration in the differentiation and maintenance of the structural integrity of the trabecular meshwork. The loss of structural support of the buphthalmic trabecular meshwork may be a factor in the wide variation in intraocular pressure and may allow for compression of the trabecular meshwork against the aqueous plexus.

Osteoclasts isolated from membranous bone in children exhibit nuclear estrogen and progesterone receptors.

Osteoclasts were isolated from membranous bone from four children without metabolic bone disease who were undergoing craniotomy for either tumor or trauma. Both freshly isolated osteoclasts and those cultured for 4-7 days exhibited the following characteristics: production of tartrate-resistant acid phosphatase (9.5-14.8 units), contraction in response to application of 100 mg/ml of human calcitonin, and formation of resorption lacunae on devitalized bone wafers. Nuclear estrogen and progesterone receptors were demonstrated by immunohistochemical techniques and quantitated in two of the patients by radioimmunoassay (estrogen receptor RIA, 23.6 and 23.8 cpm/micrograms protein; progesterone receptor RIA, 36.7 and 74.2 cpm/micrograms protein). The demonstration of sex steroid hormone receptors in the nucleus of osteoclasts derived from children with normal membranous bone has established a potential mechanism whereby direct modulation of bone resorption by the sex steroid estrogen and progesterone may occur.

Sex steroid hormone receptors in normal and dysplastic bone disorders in children.

Children with monostotic and polyostotic bone dysplasias often exhibit localized bone overgrowth. We investigated the presence of nuclear estrogen and nuclear progesterone receptors by solid-phase radioimmunoassay, immunocytochemistry, and radioligand binding in osteoblast cell cultures derived from the areas of overgrowth of membranous bone, noninvolved membranous bone, and normal membranous bone from children undergoing elective craniotomy. Membranous bone of normal children had demonstrable levels of nuclear estrogen and progesterone receptors identified by radioimmunoassay and immunocytochemical assay. Two- to threefold increased levels of these receptors (p less than 0.001 versus normals) were found in cultures derived from the involved bone of two children with monostotic fibrous dysplasia and in one patient with polyostotic dysplasia (McCune-Albright syndrome). The noninvolved bone in our patients with fibrous dysplasia exhibited nuclear sex steroid hormone receptor levels similar to those in the normal children. Radioligand binding studies demonstrated increased sex steroid hormone receptors in cultures derived from involved osteoblasts. The presence of an increased level of sex steroid hormone receptor was accompanied by increased alkaline phosphatase activity and increased production of osteocalcin in vitro compared to normal or noninvolved bone. The mechanisms by which sex steroid hormone receptor levels are increased in the ostotic dysplasias remain to be established.

Topical ocular anesthetics affect epithelial cytoskeletal proteins of wounded cornea.

The purpose of this study was to characterize by immunofluorescent microscopy, the cytoskeletal proteins actin and myosin and the protein calmodulin (CaM) in trephined, n-heptanol treated murine corneal epithelium during in vitro organ culture before and following topical ocular anesthetic application. The study has shown that corneal epithelial wounds exposed to anesthetic agents fail to close, even after 24 hr of culture. Failure of the wound to close was accompanied by a general decrease in immunofluorescence for actin myosin and CaM following application of the anesthetics.

Vitamin A-induced suppression/enhancement of protein glycosylation and neurulation.

Glycoconjugates play major roles in many cellular functions, e.g. cell migration and cell-to-cell adherence, which are involved in neurulation. The maternal administration of vitamin A on gestation day 8.5 and 9.0 resulted in a high percentage of primary and secondary neurulation defects in gestation day 12 mouse embryos. The neuroepithelium of normal and abnormal embryos was analyzed by one- and two-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis and one-dimensional Western blots using concanavalin A (Con A) and peroxidase-conjugated wheat germ agglutinin (WGA) lectins. In vitamin A abnormal embryos, WGA binding was decreased to glycoproteins with apparent molecular weights of 15,000 and 30,000 daltons on Western blots, whereas in vitamin A normal embryos, WGA binding was increased to these glycoproteins on Western blots. Computer-aided fluorescence microscopy using fluorescein isothiocyanate (FITC)-conjugated lectins on 1-micron araldite plastic sections indicated a decrease in FITC-WGA binding to the free surface of nonneurulated neuroepithelium. These results suggest: (1) vitamin A administration may have induced a suppression of WGA-binding carbohydrate residues on 15,000- and 30,000-dalton glycoproteins in abnormal embryos, and (2) modification in the type, amount, and distribution of glycoconjugates may provide a basis for the cellular mechanisms of abnormal development of the neural tube.

Spectrin subtypes in mammalian brain: an immunoelectron microscopic study.

Spectrin is a major cytoskeletal component of the brain. At least 2 distinct spectrin subtypes are found in mammalian brain: brain spectrin(240/235) and brain spectrin(240/235E). In the present study spectrin subtypes were localized in the adult mouse brain by immunoelectron microscopy using antibodies that recognize each subtype. Brain spectrin(240/235E) was concentrated in neuronal cell bodies, dendrites, and postsynaptic terminals. It was also prominently associated with the plasma membrane, microtubules, filaments, mitochondria, endoplasmic reticulum, and nuclear envelope, and it appeared to interconnect structural elements within the cell. Brain spectrin(240/235E) also was localized to the plasma membrane, nuclear envelope, and cytoplasmic organelles of glial cell bodies. Brain spectrin(240/235) was detected in axons and presynaptic elements, where it was associated with the plasma membrane, microtubules, filaments, synaptic vesicles, and mitochondria. These results show that spectrin is distributed throughout the cytoplasm of neural cells, the location of spectrin is dependent on subtype, and the cytoplasmic surface of plasma membrane and organelles contains an extensive and intricate spectrin meshwork.

Actinlike material in Pseudomonas aeruginosa.

Actinlike material was obtained from disrupted Pseudomonas aeruginosa cells by a modification of the method of Hancock and Nikaido (J. Bacteriol. 136:381-390) for isolating outer membrane vesicles. Pelleted membranes were dissolved in Laemmli sample buffer and electrophoresed in parallel lanes with purified rabbit skeletal muscle actin. The bacterial preparation migrated similarly to rabbit skeletal muscle actin. A doublet band was detectable by affinity-purified antiactin antibody in a passive transfer immunoblot. Molecular weight of the actinlike protein doublet was 60,000 to 63,000 as determined by linear regression analysis of Bio-Rad low-molecular-weight standards run on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Transmission electron microscopy of the actinlike material in high salt concentrations revealed 10- to 14-nm filaments of various lengths. Despite its ability to form filaments and to react with a polyclonal rabbit antiactin antibody, the bacterial filaments did not bind the S-1 fragment of heavy meromyosin.

Change with age in murine corneal epithelial actin and myosin: immunofluorescent and ELISA analyses.

The purpose of this study was to use immunofluorescence and ELISA immunoassay to determine whether the cellular distribution and concentration of corneal epithelial actin and myosin change with chronologic age. Diffuse anti-actin and anti-myosin indirect immunofluorescence was observed within the cytoplasm of the corneal epithelium from mice aged postnatal day (PND) 1-18 months. Additionally, highly fluorescent punctate foci were first observed in cortical cytoplasm consistently for both anti-actin and anti-myosin at PND 14. This fluorescent pattern remained relatively unchanged for the remaining ages examined. An enzyme-linked immunosorbent assay (ELISA) method was used to quantitate the amount of actin and myosin in corneal epithelium from mice aged PND 1 to 24 months. Corneal epithelial sheets were removed from whole eyes and processed for ELISA assay. Actin cellular concentration increased from PND 1-7 and decreased from PND 7-16. These results were statistically significant (p less than .005). No statistically significant difference in actin concentration was found for any of the remaining ages examined (PND 16-24 months). Myosin concentration increased from PND 1-7 and decreased until PND 14. These results also were statistically significant (p less than .005 and p less than .005, respectively). There was no significant change in myosin concentration for any of the remaining ages examined (PND 16-24 months).

Actin filament localization and distribution in the young adult mouse cornea: a correlative immunofluorescent and cytochemical study.

The localization and distribution of actin filaments was determined in the corneal epithelium and endothelium of the young adult Swiss Webster mouse by correlative indirect immunofluorescence using rabbit anti-skeletal muscle actin antiserum and by in situ labelling with heavy meromyosin subfragment-1 (HMM-S1). A diffuse fluorescent staining was observed in the cytoplasm of epithelial and endothelial cells and in stromal keratocytes. In addition, a highly fluorescent punctate cytoplasmic staining was seen only in the corneal epithelium. Ultrastructurally, HMM-S1 decorated actin filament bundles were generally distributed within the cytoplasm of glycerinated superficial, wing and basal epithelial cells and within pedicle-like processes on the posterior surface of superficial and wing cells. Actin filament bundles also were seen in close association with intermediate filament bundles in the epithelium. HMM-S1 labelled filaments were observed in the endothelial cytoplasm anteriorly near Descemet's membrane, posteriorly near the anterior chamber and adjacent to lateral cell borders. Actin filaments in epithelial and endothelial cells appeared to insert into the plasma membrane.