Pig eye trabeculectomy-a wet-lab teaching model.

PURPOSE: A teaching model for trabeculectomy is described using pig eyes prepared in formalin. METHOD: The model enables trainee surgeons to practice various aspects of tissue handling required for successful trabeculectomy including the construction of a fornix-based conjunctival flap, scleral flap with buried releasable sutures, and water-tight conjunctival closure. RESULTS: Exposure to the necessary skills required to perform trabeculectomy surgery can be improved by the use of wet laboratory practice. CONCLUSIONS: Trabeculectomy surgery experience is becoming more limited as fewer procedures are being performed due to the efficacy of recent medications. Wet laboratories will become an increasingly important aspect of a comprehensive ophthalmology training programme.

New polyoxygenated briarane diterpenoids, briaexcavatolides O-R, from the gorgonian Briareum excavatum.

Four new polyoxygenated briarane-type diterpenoids, briaexcavatolides O-R (1-4), have been isolated from a gorgonian octocoral Briareum excavatum. Their structures were determined using spectroscopic and chemical methods. Metabolites 1-3 were found to contain oxygenated substituents at C-2, C-3, and C-4, and the relative configurations were assigned as 2R*,3R*,4R* at these three positions. Briaranes containing this type of stereochemistry are reported for the first time. The structures of metabolites 1 and 2 were further confirmed by single-crystal X-ray analyses. Compound 2 has been shown to exhibit significant cytotoxicity toward P-388 and HT-29 cancer cells.

Role for transcription Pax5A factor in maintaining commitment to the B cell lineage by selective inhibition of granulocyte-macrophage colony-stimulating factor receptor expression.

During early B lymphopoiesis, developing B cells maintain lineage commitment despite the local presence of myeloid lineage-promoting cytokines such as GM-CSF and IL-3. Previous observations suggest that the B cell-specific transcription factor Pax5A (paired box 5A transcription factor) plays a role in maintaining B cell lineage commitment by limiting expansion and survival of early IL-3/GM-CSF-dependent myeloid lineage cells. To define a mechanism by which Pax5A can exert these inhibitory effects on myeloid lineage differentiation, an inducible form of the Pax5A protein was expressed in the myeloid cell line FDC-P1. This cell line models myeloid progenitors in that it responds to the survival and growth-potentiating effects of IL-3 and GM-CSF. We observed that enforced expression of Pax5A selectively suppressed proliferation in response to GM-CSF, but not IL-3. This effect was associated with specific down-regulation of GM-CSFR alpha-chain, but not beta-chain expression. These data provide a molecular mechanism to enforce commitment to the B cell lineage despite the presence of GM-CSF, a factor that has been shown to convert early developing B cells to the myeloid lineage. Furthermore, they indicate a role for B cell Pax5A expression in maintaining rather than directing commitment to the B cell lineage.

Dichloro(2,2'-diamino-4,4'-bi-1,3-thiazole-N3,N3')copper(II).

The title complex, [CuCl(2)(C(6)H(6)N(4)S(2))], has a flattened tetrahedral coordination. The Cu(II) atom is located on a twofold rotation axis and is coordinated by two N atoms from a chelating 2,2'-diamino-4,4'-bi-1,3-thiazole ligand and by two Cl atoms. Intramolecular hydrogen bonding exists between the amino groups of the 2,2'-diamino-4,4'-bi-1,3-thiazole ligand and the Cl atoms. The intermolecular separation of 3.425 (1) A between parallel bithiazole rings suggests there is a pi-pi interaction between them.

Briaexcavatolides K-N, new briarane diterpenes from the gorgonian Briareum excavatum.

Four new briarane diterpenes, briaexcavatolides K-N (1-4), along with a known diterpene, 5, have been isolated from the Taiwanese gorgonian Briareum excavatum. The structures of the new metabolites were established by extensive spectral analyses. Furthermore, the structure, including the relative configuration of briaexcavatolide K (1), was confirmed by a single-crystal X-ray analysis. Briaexcavatolides K and L (1 and 2) are the only briarane diterpenes known to possess hydroxyl groups at the C-8beta and C-17alpha positions, respectively. Cytotoxicity of these metabolites toward various cancer cell lines also is described.

Junceellolides E-G, new briarane diterpenes from the West Pacific Ocean gorgonian Junceella fragilis.

Three new diterpenes, junceellolides E-G (1-3), along with an unnamed known diterpene 4, possessing the briarane carbon skeleton, have been isolated from the gorgonian Junceella fragilis collected in Taiwanese tropical waters. The structures of the new metabolites 1-3 were elucidated by extensive spectral analyses. The six-membered rings in junceellolides E and F (1 and 2) were found to exist in boat conformations. The structure, including the relative configuration of junceellolide E (1) was further confirmed by a single-crystal X-ray analysis.

The orphan nuclear receptor Tlx regulates Pax2 and is essential for vision.

Although the development of the vertebrate eye is well described, the number of transcription factors known to be key to this process is still limited. The localized expression of the orphan nuclear receptor Tlx in the optic cup and discrete parts of the central nervous system suggested the possible role of Tlx in the formation or function of these structures. Analyses of Tlx targeted mice revealed that, in addition to the central nervous system cortical defects, lack of Tlx function results in progressive retinal and optic nerve degeneration with associated blindness. An extensive screen of Tlx-positive and Tlx-negative P19 neural precursors identified Pax2 as a candidate target gene. This identification is significant, because Pax2 is known to be involved in retinal development in both the human and the mouse eye. We find that Pax2 is a direct target and that the Tlx binding site in its promoter is conserved between mouse and human. These studies show that Tlx is a key component of retinal development and vision and an upstream regulator of the Pax2 signaling cascade.

Lysozyme association with nucleic acids.

Lysozyme is well known for the ability to hydrolyze the cell wall of bacteria. Based on the similarity of structure between lysozyme and histones as seen from the results of X-ray crystal structure determinations, we have postulated that binding to nucleic acids may be another biological function of lysozyme. We have therefore begun a systematic study of the interactions of lysozyme and related molecules with nucleic acids, and present here a preliminary report. Binding to DNA and RNA has been demonstrated from gel electrophoresis, enzyme activity, and coprecipitation studies. We suggest that this function of lysozyme will provide an explanation why Lee-Huang et al. (1999) [Proc. Natl. Acad. Sci. USA 96, 2678-2681] were able to call lysozyme a "killer protein" against the AIDS virus, and may provide a new avenue of research on AIDS therapy.

Cytotoxic terpenoids from the Formosan soft coral Nephthea brassica.

Two new cytotoxic cembranoid diterpenes, brassicolide (1) and brassicolide acetate (2); a new cytotoxic sesquiterpene, (-)-4alpha-O-acetyl-selin-11-en (3); and six cytotoxic terpenoids, (-)-selin-11-en-4alpha-ol (4), 2-hydroxynephthenol (5), nephthenol (6), cembrene A (7), epoxycembrene A (8), and (-)-beta-elemene (9), have been isolated from the Formosan soft coral Nephthea brassica. The structures of compounds 1-9 were determined by spectral, chemical, and X-ray crystallographic analysis.

BSAP/Pax5A expression blocks survival and expansion of early myeloid cells implicating its involvement in maintaining commitment to the B-lymphocyte lineage.

Early B lymphopoiesis is marked by plasticity between the myeloid and B lineages. An attractive model for B-lineage development is that commitment to this lineage is partly determined by the ordered expression of genes that prohibit switching to the myeloid lineage. In this regard, whereas the role of the B-cell-specific transcription factor BSAP/Pax5A in regulating B-lymphoid-restricted gene expression has been well-established, its role in maintaining B-lineage commitment is unclear. Thus, BSAP/Pax5A was constitutively expressed in the multipotent EML cell line, which can be directed toward the myeloid lineage by culture with interleukin-3 (IL-3) and retinoic acid. EML cells expressing BSAP/Pax5A successfully acquired the myeloid lineage markers CD11b and F4/80 in response to IL-3 and retinoic acid, indicating differentiation to the myeloid lineage. However, these early myeloid cells failed to expand in culture with granulocyte-macrophage colony-stimulating factor and were directed instead toward an apoptotic pathway. In parallel, primary bone marrow stem cells transduced with retrovirus constitutively expressing BSAP/Pax5A began myeloid cell differentiation, but like the transformed EML model failed to expand in response to myeloid growth factors. These studies identify a role for BSAP/Pax5A in suppressing the response to myeloid growth factors, which may be a component of the regulatory processes that limit plasticity of early B-lymphoid progenitors.

Molecular structure of the amyloid-forming protein kappa I Bre.

The molecular structure of the amyloid-forming Bence-Jones protein kappa I Bre has been determined by X-ray crystallography at 2.0 A resolution. The fragment from the kappa chain of immunoprotein contains 107 amino acid residues, and polymerizes in the crystal form into a giant helical spiral, surrounding a cylinder of water 50 A in diameter with a repeat of 77.56 A, containing 12 kappa molecules, plus another 12 molecules from neighboring parallel spirals. The resulting structure has many features which have been found or suggested from studies on the protein fibrils found in amyloid deposits. From the results of the X-ray crystal structure a hypothesis is presented for the structure and formation of the amyloid fibril.

Novel cytotoxic diterpenes, excavatolides A-E, isolated from the Formosan gorgonian Briareum excavatum.

The chemistry of Briareum excavatum, a Formosan gorgonian coral, was investigated. This study has led to the isolation of five novel marine natural products, excavatolides A-E (1-5), together with brianolide (6). The structures of the above compounds were established by spectral and chemical methods. The relative configuration of excavatolide B (2) was further confirmed by a single-crystal X-ray structure analysis. Cytotoxicity of these compounds toward various cancer cell lines also is described.

Novel cytotoxic cembranoids from the soft coral Sinularia flexibilis.

Three new cytotoxic cembranoid diterpenes, sinuflexolide (1), dihydrosinuflexolide (2), and sinuflexibilin (3), have been isolated from the soft coral Sinularia flexibilis. The structures of compounds 1-3 were determined by spectral and X-ray crystallographic analysis.

An essential role for retinoid receptors RARbeta and RXRgamma in long-term potentiation and depression.

Hippocampal long-term potentiation (LTP) and long-term depression (LTD) are the most widely studied forms of synaptic plasticity thought to underlie spatial learning and memory. We report here that RARbeta deficiency in mice virtually eliminates hippocampal CA1 LTP and LTD. It also results in substantial performance deficits in spatial learning and memory tasks. Surprisingly, RXRgamma null mice exhibit a distinct phenotype in which LTD is lost whereas LTP is normal. Thus, while retinoid receptors contribute to both LTP and LTD, they do so in different ways. These findings not only genetically uncouple LTP and LTD but also reveal a novel and unexpected role for vitamin A in higher cognitive functions.

Expression of human group II PLA2 in transgenic mice results in epidermal hyperplasia in the absence of inflammatory infiltrate.

Group II PLA2 has been implicated in inflammatory processes in both man and other animals and has been shown to be involved in inflammatory conditions, such as arthritis and sepsis. Transgenic mice expressing the human group II PLA2 gene have been generated using a 6.2-kb genomic fragment. These mice express the group II PLA2 gene abundantly in liver, lung, kidney, and skin, and have serum PLA2 activity levels approximately eightfold higher than nontransgenic littermates. The group II PLA2 transgenic mice reported here exhibit epidermal and adnexal hyperplasia, hyperkeratosis, and almost total alopecia. The chronic epidermal hyperplasia and hyperkeratosis seen in these mice is similar to that seen in a variety of dermatopathies, including psoriasis. However, unlike what is seen with these dermatopathies, no significant inflammatory-cell influx was observed in the skin of these animals, or in any other tissue examined. These mice provide an important tool for examining group II PLA2 expression, and for determining the role of group II PLA2 in normal and disease physiology. They serve as an in vivo model for identifying inhibitors of group II PLA2 activity and gene expression.

Sinugibberol, a new cytotoxic cembranoid diterpene from the soft coral Sinularia gibberosa.

Bioactivity-guided fractionation of a chloroform extract of the soft coral Simularia gibberosa afforded a new cytotoxic cembranoid diterpene, sinugibberol [1]. The structure of 1 was determined by spectral and X-ray crystallographic analysis.

Sequence specific inhibition of human type II phospholipase A2 enzyme activity by phosphorothioate oligonucleotides.

Phosphorothioate oligonucleotides were identified which directly inhibited human type II phospholipase A2 (PLA2) enzyme activity in a sequence specific manner. The minimum pharmacophore common to all oligonucleotides which inhibited PLA2 enzyme activity consisted of two sets of three or more consecutive guanosine residues in a row. These oligonucleotides appear to form G quartets resulting in the formation of oligonucleotide aggregates. Additionally, a phosphorothioate backbone was required to be effective inhibitors of type II PLA2. The activity of one oligodeoxynucleotide, IP 3196 (5'-GGGTGGGTATAGAAGGGCTCC-3') has been characterized in more detail. IP 3196 inhibited PLA2 enzyme activity when the substrate was presented in the form of a phospholipid bilayer but not when presented in the form of a mixed micelle with anionic detergents. Human type II PLA2 was 50-fold more sensitive to inhibition by IP 3196 than venom and pancreatic type I enzymes. These data demonstrate that phosphorothioate oligonucleotides can specifically inhibit human type II PLA2 enzyme activity in a sequence specific manner.

Inhibition of endothelial cell adhesion molecule expression with antisense oligonucleotides.

In response to inflammatory stimuli, expression of a group of proteins that bind circulating leukocytes (endothelial-leukocyte adhesion molecules) are induced on the luminal surface of vascular endothelium. A series of phosphorothioate oligonucleotides 18 to 21 bases in length were designed and synthesized to hybridize selectively to the mRNA, which encodes three such endothelial-leukocyte adhesion molecules; human intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), and E-selectin. Antisense oligonucleotides were identified that selectively inhibited ICAM-1, VCAM-1, and E-selectin expression in HUVEC. Oligonucleotides that hybridized to the 3'-untranslated region of either ICAM-1, VCAM-1, or E-selectin mRNAs promoted a selective reduction in the respective mRNA levels. In contrast, oligonucleotides that hybridized to 5'-untranslated sequences did not significantly reduce target mRNA levels, although they did promote a reduction in protein expression. With the use of flow cytometry to measure cell surface expression, ICAM-1 and E-selectin were selectively inhibited by their respective antisense oligonucleotide. At low concentrations of oligonucleotides, only VCAM-1 antisense oligonucleotides inhibited VCAM-1 expression. However, at an oligonucleotide concentration of 50 nM or greater, phosphorothioate oligonucleotides not predicted to hybridize to VCAM-1 mRNA also reduced VCAM-1 expression. The sequence-independent inhibition of VCAM-1 expression by phosphorothioate oligonucleotides could be the result of a perturbation in the transcriptional regulation of the VCAM-1 gene. ICAM-1, VCAM-1, and E-selectin antisense oligonucleotides reduced adhesion of HL-60 cells to TNF-activated HUVEC. These data demonstrate that phosphorothioate oligonucleotides are capable of selectively inhibiting the expression of ICAM-1, VCAM-1, and E-selectin in HUVEC.