Nuclear Phosphoinositides: Their Regulation and Roles in Nuclear Functions.

Polyphosphoinositides (PPIns) are a family of seven lipid messengers that regulate a vast array of signalling pathways to control cell proliferation, migration, survival and differentiation. PPIns are differentially present in various sub-cellular compartments and, through the recruitment and regulation of specific proteins, are key regulators of compartment identity and function. Phosphoinositides and the enzymes that synthesise and degrade them are also present in the nuclear membrane and in nuclear membraneless compartments such as nuclear speckles. Here we discuss how PPIns in the nucleus are modulated in response to external cues and how they function to control downstream signalling. Finally we suggest a role for nuclear PPIns in liquid phase separations that are involved in the formation of membraneless compartments within the nucleus.

Orientation selectivity of cortical neurons during intracellular blockade of inhibition.

Neurons in the primary visual cortex of the cat are selectively activated by stimuli with particular orientations. This selectivity can be disrupted by the application of antagonists of the inhibitory neurotransmitter gamma-aminobutyric acid (GABA) to a local region of the cortex. In order to determine whether inhibitory inputs are necessary for a single cortical neuron to show orientation selectivity, GABA receptors were blocked intracellularly during whole cell recording. Although the membrane potential, spontaneous activity, subfield antagonism, and directional selectivity of neurons were altered after they were perfused internally with the blocking solution, 18 out of 18 neurons remained selective for stimulus orientation. These results indicate that excitatory inputs are sufficient to generate orientation selectivity.

Orientation maps of subjective contours in visual cortex.

Responses to subjective contours in visual cortical areas V1 and V2 in adult cats were investigated by optical imaging of intrinsic signals and single-unit recording. Both V1 and V2 contain maps of the orientation of subjective gratings that have their basis in specific kinds of neuronal responses to subjective orientations. A greater proportion of neurons in V2 than in V1 show a robust response to subjective edges. Through the use of subjective stimuli in which the orientation of the luminance component is invariant, an unmasked V1 response to subjective edges alone can be demonstrated. The data indicate that the processing of subjective contours begins as early as V1 and continues progressively in higher cortical areas.

Changing objects lead briefly flashed ones.

Continuous, predictable events and spontaneous events may coincide in the visual environment. For a continuously moving object, the brain compensates for delays in transmission between a retinal event and neural responses in higher visual areas. Here we show that it similarly compensated for other smoothly changing features. A disk was flashed briefly during the presentation of another disk of continuously changing color, and observers compared the colors of the disks at the moment of flash. We also tested luminance, spatial frequency and pattern entropy; for all features, the continuously changing item led the flashed item in feature space. Thus the visual system's ability to compensate for delays in information about a continuously changing stimulus may extend to all features. We propose a model based on backward masking and priming to explain the phenomenon.

Tight junction biogenesis in the early Xenopus embryo.

Tight junctions (TJs) perform a critical role in the transport functions and morphogenetic activity of the primary epithelium formed during Xenopus cleavage. Biogenesis of these junctions was studied by immunolocalization of TJ-associated proteins (cingulin, ZO-1 and occludin) and by an in vivo biotin diffusion assay. Using fertilized eggs synchronized during the first division cycle, we found that membrane assembly of the TJ initiated at the animal pole towards the end of zygote cytokinesis and involved sequential incorporation of components in the order cingulin, ZO-1 and occludin. The three constituents appeared to be recruited from maternal stores and were targeted to the nascent TJ site by different pathways. TJ protein assembly was focused precisely to the border between the oolemma-derived apical membrane and newly-inserted basolateral membrane generated during cytokinesis and culminated in the formation of functional TJs in the two-cell embryo, which maintained a diffusion barrier. New membrane formation and the generation of cell surface polarity therefore precede initiation of TJ formation. Moreover, assembly of TJ marker protein precisely at the apical-basolateral membrane boundary was preserved in the complete absence of intercellular contacts and adhesion. Thus, the mechanism of TJ biogenesis in the Xenopus early embryo relies on intrinsic cues of a cell autonomous mechanism. These data reveal a distinction between Xenopus and mammalian early embryos in the origin and mechanisms of epithelial cell polarization and TJ formation during cleavage of the egg.

Differentiation of the epithelial apical junctional complex during mouse preimplantation development: a role for rab13 in the early maturation of the tight junction.

We have investigated the mechanisms by which the epithelial apicolateral junctional complex (AJC) is generated during trophectoderm differentiation in the mouse blastocyst using molecular, structural and functional analyses. The mature AJC comprises an apical tight junction (TJ), responsible for intercellular sealing and blastocoel formation, and subjacent zonula adherens E-cadherin/catenin adhesion complex which also extends along lateral membrane contact sites. Dual labelling confocal microscopy revealed that the AJC derived from a single 'intermediate' complex formed following embryo compaction at the 8-cell stage in which the TJ-associated peripheral membrane protein, ZO-1alpha- isoform, was co-localized with both alpha- and beta-catenin. However, following assembly of the TJ transmembrane protein, occludin, from the early 32-cell stage when blastocoel formation begins, ZO-1alpha- and other TJ proteins (ZO-1alpha+ isoform, occludin, cingulin) co-localized in an apical TJ which was separate from a subjacent E-cadherin/catenin zonula adherens complex. Thin-section electron microscopy confirmed that a single zonula adherens-like junctional complex present at the AJC site following compaction matured into a dual TJ and zonula adherens complex at the blastocyst stage. Embryo incubation in the tracer FITC-dextran 4 kDa showed that a functional TJ seal was established coincident with blastocoel formation. We also found that rab13, a small GTPase previously localized to the TJ, is expressed at all stages of preimplantation development and relocates from the cytoplasm to the site of AJC biogenesis from compaction onwards with rab13 and ZO-1alpha- co-localizing precisely. Our data indicate that the segregation of the two elements of the AJC occurs late in trophectoderm differentiation and likely has functional importance in blastocyst formation. Moreover, we propose a role for rab13 in the specification of the AJC site and the formation and segregation of the TJ.

Dysplasia epiphysealis hemimelica of the talus: two case reports.

Trevor's disease, also known as dysplasia epiphysealis hemimelica, is an uncommon skeletal developmental disorder representing an osteochondroma occurring in one or more epiphyses. We present 2 cases of dysplasia epiphysealis hemimelica in an 8-year-old female and a 12-year-old male who suffered from a hard swelling of the ankle joint. The female patient was treated by surgical excision and the male patient conservatively.

The solution conformations of the subclasses of human IgG deduced from sedimentation and small angle X-ray scattering studies.

The solution conformations of human immunoglobulin G subclass molecules have been investigated by sedimentation and small-angle X-ray scattering techniques. Both methods qualitatively indicate IgG3 to be an extended molecule relative to IgG1. Sedimentation data have been collected for a number of paraproteins of all four subclasses and the hinge-deleted IgG1Dob protein. The known crystal structure of Dob allows the use of this protein as a basis for the proposal of models of the average conformations of IgG subclasses which are consistent with experimental s(0)20,w values. IgG1 is suggested to have a hinge length of 0-15 A and non-coplanar Fab arms; IgG2 to be effectively hingeless with folded-back Fab arms; IgG3 to have an extended hinge of the order of 100 A and IgG4 to be effectively hingeless and T-shaped. The possible correlation of these conformations with subclass function is discussed.

Cell adhesion in the preimplantation mammalian embryo and its role in trophectoderm differentiation and blastocyst morphogenesis.

Cell adhesion plays a critical role in the differentiation of the trophectoderm epithelium and the morphogenesis of the blastocyst. In the mouse embryo, E-cadherin mediated adhesion initiates at compaction at the 8-cell stage, regulated post-translationally via protein kinase C and other signalling molecules. E-cadherin adhesion organises epithelial polarisation of blastomeres at compaction. Subsequently, the proteins of the epithelial tight junction are expressed and assemble at the apicolateral contact region between outer blastomeres in three phases, culminating at the 32-cell stage when blastocoel cavitation begins. Cell adhesion events also coordinate the cellular allocation and spatial segregation of the inner cell mass (ICM) of the blastocyst, and the maintenance of epithelial (trophectoderm) and non-epithelial (ICM) phenotypes during early morphogenesis.

Junctional complexes in the early mammalian embryo.

Preimplantation embryos generate intercellular junctions during differentiation of the trophectoderm epithelium and the formation of the blastocyst. These membrane complexes comprise gap junctions, adherens junctions, tight junctions, and desmosomes, each performing fundamental roles in cellular communication, adhesion, and differentiation. The mouse embryo has been used as a model for the biogenesis of cell junctions. Their construction is achieved by temporally regulated gene expression programs. Mechanisms of junction membrane assembly include the timing of transcription, translation, and post-translational modifications of specific junctional proteins. Human embryos exhibit similar expression programs, and defects in these programs may contribute to reduced embryo viability.

Autosomal dominant iris hypoplasia is caused by a mutation in the Rieger syndrome (RIEG/PITX2) gene.

PURPOSE: To determine whether autosomal dominant iris hypoplasia is caused by mutations in the newly described gene for Rieger syndrome (RIEG/PITX2). METHOD: Mutation screening and sequence analysis was performed in a single family. RESULTS: A novel mutation in the RIEG/PITX2 gene was found in all affected but no unaffected individuals. This mutation would be expected to result in an arginine to tryptophan amino acid change in the homeodomain of solurshin, the RIEG/ITX2 gene product. CONCLUSION: Autosomal dominant iris hypoplasia is caused by a defect in the same gene that is defective in many cases of Rieger syndrome.

Compression of space in visual memory.

Human observers had to point to the location of a briefly presented target by means of a mouse after a brief delay following target offset. It was found that observers systematically mislocalized the target closer to the center of gaze, and to visually salient markers in the visual display. A perceptual judgment task revealed that these errors in localization were independent of whether or not eye movements were made, and even of planning for them, thereby demonstrating that the effect was a perceptual phenomenon, not a sensorimotor one. Further experiments demonstrated clearly that the magnitude of the time interval between target presentation and judgment regarding its spatial location was the critical parameter. A longer time interval between the event and its report enhanced significantly the amplitude of compression, thus establishing this phenomenon as a visual memory effect. We conclude that visual memory of spatial location is distorted over time in a systematic, monotonic fashion as a result of the sustained fixation of the observer on a fixed location during and shortly after target presentation, or by the continual presence of stable, salient landmarks in the environment.

Influence of an optimized non-ionic emulsifier blend on properties of oil-in-water emulsions.

Properties of oil-in-water emulsions containing non-ionic emulsifiers were evaluated in relation to nature of the dispersed phase, emulsifier composition and processing parameters. Particle size of mineral oil (hydrocarbons)-in-water emulsions was independent of the HLB of an optimized emulsifier blend, whereas, the particle size of olive oil (triglycerides)-in-water emulsions was the smallest at the optimum HLB of the emulsifier blend. The non-ionic emulsifiers reduced the particle size of mineral oil emulsions more efficiently than that of olive oil emulsions. Contrary to previously published reports, the nature of the dispersed phase, HLB of the emulsifier blend or the initial particle size of emulsions showed no influence on the final particle stability of the emulsions. This difference was attributed to the optimization of the emulsifier blend and processing parameters in the preparation of emulsions.

Effect of polymers on dissolution from drug suspensions.

The effect of three viscosity grades of methylcellulose on the dissolution-dialysis rate of nitrofurantoin suspensions was investigated using a cell designed to provide a large surface area for dialysis. Apparent dialytic rate constants of drug dispersions and solutions were measured in 0.1 N HC1 and in pH 7.4 buffer. Samples containing methylcellulose had lower rates of dialysis, with the lowest rate being observed for samples in which the polymer was used as the suspending agent. The reduced rate of dialysis of the drug suspension containing methylcellulose is thought to be due to complexation of the drug in solution with the polymer as well as formation of microscopic regions of high viscosity surrounding the undissolved drug particles leading to a reduction in the dissolution rate of the drug. An empirical relationship was obtained to enable the estimation of the effective drug concentration in the dissolution chamber for drug dispersions. The method is based on utilizing dialysis rate data of drug solutions. This relationship could be used for comparing suspension formulations in terms of the amount of drug available for dialysis.

Evaluation of adsorption from dispersion charge profile.

The evaluation of adsorption behavior from the charge profile of an adsorbent dispersion determined as a function of the adsorbate concentration was investigated by the streaming current measurement, using methylene blue and erythrosine as adsorbates and activated charcoal, microcrystalline cellulose, and polyvinylpolypyrrolidone as adsorbents. Adsorption capacity was evaluated using streaming current versus solute concentration plots of dye solutions and of corresponding dye solutions equilibrated with the adsorbent. It was also determined by adsorption isotherm measurement and application of the Langmuir equation. Good agreement was obtained between the adsorption capacity values from streaming current data and adsorption isotherm measurements for microcrystalline cellulose, suggesting that adsorption here was a surface phenomenon without water-soluble extractives affecting particle charge. Similar agreement was not obtained for charcoal adsorption, and this result was attributed to the unusual adsorption behavior of charcoal. Since polyvinylpolypyrrolidone gave a dispersion with high particle charge, the streaming current method could not be used with this system. The charge profile could be useful in characterizing powders for unusual adsorption behavior and possible water-soluble extractives as well as for surface area estimation in the absence of these factors.

Reduced dialysis of nitrofurantoin and hydrocortisone acetate from methylcellulose solutions.

Factors affecting reduction in the permeation rate of nitrofurantoin and hydrocortisone acetate from methylcellulose solutions through a cellulose membrane were investigated. The factors evaluated were the effect of polymer agglomeration and whether the drugs formed a complex with the polymer. The influence of polymer agglomeration was evaluated by dynamic dialysis studies of nitrofurantoin dissolved in solutions of methylcellulose 400 cps, which agglomerates, and in methylcellulose 50 cps, which does not exhibit this behavior. Dialysis rates of this drug were measured as a function of polymer concentration, ionic strength, heating time prior to measurement, and temperature. The effect of polymer agglomeration on the permeation rates of hydrocortisone acetate was studied by varying the ionic strength of the drug-polymer solution. Complex formation studies were conducted for both drugs by the equilibrium dialysis method. Polymer agglomeration caused a substantial reduction in the dialysis rate of both drugs in methylcellulose 400 cps solutions. There was less reduction in the dialysis rate of nitrofurantoin with increased polymer concentration of methylcellulose 50 cps solutions, which was attributed to the increase in the bulk viscosity of the solution. There was no evidence of complex formation of the drugs with the polymers.

Lactoferrin cDNA. Expression and in vitro mutagenesis.

The full length copy DNA (cDNA) for human lactoferrin has been synthesised by the polymerase chain reaction (PCR) using sequence specific primers. The template was first strand cDNA, synthesised from human bone marrow RNA using oligo(dT) to prime DNA synthesis by MMLV reverse transcriptase. The full-length human lactoferrin cDNA has been expressed in baby hamster kidney (BHK) cells using the expression vector pNUT. The protein expressed from the cloned cDNA is secreted into the culture medium and yields of up to 40 mg per litre have been obtained. A mutant protein corresponding to the N-lobe of human lactoferrin (LfN) has also been expressed in BHK cells. The cDNA coding for this protein was produced by the introduction of stop codons into the region of the cDNA corresponding to the helix linking the N- and C-lobes of the native protein. LfN is also expressed as a secreted protein and has been obtained in high yield. LfN binds iron and has UV/Vis and ESR spectra which are virtually identical to the native protein. However, the pH at which iron is released from LfN is quite different to the pH of iron release from the native and the full-length recombinant protein. A number of mutations have been introduced into LfN by site-directed mutagenesis and the mutant proteins expressed in BHK cells. These mutations involve the iron binding ligands and have been designed to introduce some of the changes found in the C-lobe of melanotransferrin into LfN. An attempt has been made to express a protein corresponding to the C-lobe of human lactoferrin (LfC) by attaching the sequence for the signal peptide of lactoferrin to the cDNA sequences coding for the C-lobe.

Combined pars plana lensectomy-vitrectomy with open-loop flexible anterior chamber intraocular lens (AC IOL) implantation for subluxated lenses.

OBJECTIVES: To review our experience with combined pars plana lensectomy-vitrectomy and open-loop flexible anterior chamber intraocular lens (AC IOL) implantation for managing subluxated crystalline lenses. METHODS: Retrospective review of 36 consecutive eyes (28 patients), all of which had subluxated crystalline lenses, managed by pars plana lensectomy-vitrectomy with insertion of an open-loop flexible AC IOL. The study was performed at the Medical College of Wisconsin, Milwaukee, over an 8-year period. RESULTS: An average preoperative visual acuity of 20/163 (range, 20/25 to hand motions) improved to 20/36 (range, 20/20 to 4/200) with surgery after a mean follow-up of 14 months (range, 1 to 59 months) (P < .001, Student's paired t test). Final visual acuity of 20/40 or better was achieved in 75% of eyes (27/36). Complications included cystoid macular edema (8% [3/36]), pupillary block (6% [2/36]), retinal detachment (3% [1/36]), hyphema (3% [1/36]), wound leak (3% [1/36]), and transient vitreous hemorrhage (3% [1/36]). No persistent ocular hypertension was seen, nor did angle abnormalities or corneal decompensation develop. CONCLUSIONS: Pars plana lensectomy-vitrectomy with AC IOL implantation appears to be an excellent technique for managing subluxated crystalline lenses. It is associated with a significant improvement in visual acuity (P < .001) and avoids many of the complications seen with extraction of a subluxated lens through a limbal wound. Additionally, use of an AC IOL offers a simplified alternative to placement of a ciliary sulcus sutured posterior chamber intraocular lens (PC IOL).

Orbital blow-out fractures: correlation of preoperative computed tomography and postoperative ocular motility.

BACKGROUND/PURPOSE: Although the management of orbital blow-out fractures was controversial for many years, refined imaging with computed tomography (CT) helped to narrow the poles of the debate. Many orbital surgeons currently recommend repair if fracture size portends late enophthalmos, or if diplopia has not substantially resolved within 2 weeks of the injury. While volumetric considerations have been generally well-served by this approach, ocular motility outcomes have been less than ideal. In one series, almost 50% of patients had residual diplopia 6 months after surgery. A fine network of fibrous septa that functionally unites the periosteum of the orbital floor, the inferior fibrofatty tissues, and the sheaths of the inferior rectus and oblique muscles was demonstrated by Koornneef. Entrapment between bone fragments of any of the components of this anatomic unit can limit ocular motility. Based on the pathogenesis of blow-out fractures, in which the fibrofatty-muscular complex is driven to varying degrees between bone fragments, some measure of soft tissue damage might be anticipated. Subsequent intrinsic fibrosis and contraction can tether globe movement, despite complete reduction of herniated orbital tissue from the fracture site. We postulated that the extent of this soft tissue damage might be estimated from preoperative imaging studies. METHODS: Study criteria included: retrievable coronal CT scans; fractures of the orbital floor without rim involvement, with or without extension into the medial wall; preoperative diplopia; surgical repair by a single surgeon; complete release of entrapped tissues; and postoperative ocular motility outcomes documented with binocular visual fields (BVFs). Thirty patients met all criteria. The CT scans and BVFs were assessed by different examiners among the authors. Fractures were classified into 3 general categories and 2 subtypes to reflect the severity of soft tissue damage within each category. "Trap-door" injuries, in which bone fragments appeared to have almost perfectly realigned, were classified as type I fractures. In the I-A subtype, no orbital tissue was visible on the sinus side of the fracture line. In the I-B subtype, soft tissue with the radiodensity of orbital fat was visible within the maxillary sinus. In type II fractures, bone fragments were distracted and soft tissue was displaced between them. In the II-A subtype, soft tissue displacement was less than, or proportional to, bone fragment distraction. In the II-B subtype, soft tissue displacement was greater than bone fragment distraction. In type III fractures, displaced bone fragments surrounded displaced soft tissue in all areas. In the III-A subtype, soft tissue and bone were moderately displaced. In the III-B subtype, both were markedly displaced. Motility outcomes were quantified by measuring the vertical excursion in BVFs. The interval between trauma and surgical repair was also determined. RESULTS: Among the 15 patients with a motility outcome in BVFs which was poorer than the median (86 degrees or less of single binocular vertical excursion), 4 patients (27%) had type A fractures; 11 patients (73%) had type B fractures. Among the 15 patients with a better outcome than the median (88 degrees or more), 10 patients (67%) had type A fractures; 5 patients (33%) had type B fractures. These differences became more defined as analysis moved away from the median. Among 5 patients with type B fractures and better than the median result in BVFs, 3 patients (60%) had surgical repair during the first week after injury. Among the 11 patients with type B fractures and less than the median result, 1 patient (9%) had repair during the first week. CONCLUSIONS: When the CT-depicted relationship between bone fragments and soft tissues is considered, a wide spectrum of injuries is subsumed under the rubric of blow-out fractures. In general, greater degrees of soft tissue incarceration or displacement, with presumably greater intrinsic damage and subsequent fibrosis, appear to result in poorer motility outcomes. Although this retrospective study does not conclusively prove its benefit, an urgent surgical approach to selected injuries should be considered.

Particle size reduction of emulsions by formulation design-II: effect of oil and surfactant concentration.

The objective of this study was to investigate the effect of the concentration of surfactant and oil on particle size reduction and stability of oil-in-water emulsion formulations containing polyhydroxy alcohols. Emulsions were prepared using an emulsifier system consisting of Tween 80 and Span 80 with 5%, 10%, 15% and 20% soybean oil and containing 50% w/w of either propylene glycol (PG) or glycerol (GLY) in the external phase. At each oil concentration, four emulsions were formulated with increasing surfactant concentration to provide emulsions with surfactant to oil (S/O) ratios of 0.1, 0.2, 0.3 and 0.4. Three parameters were evaluated, particle size reduction, particle size stability upon dilution, and viscosity. It was found that increase in S/O ratio resulted in substantial decrease in particle size in all cases. But there was a difference in the particle size reduction pattern between PG and GLY. Increase in oil concentration at the same S/O ratio caused particle size reduction for emulsions with PG but not for emulsions with GLY. The reduction in particle size was also greater for emulsions containing PG. Further, particle size of emulsions containing PG was found to be stable over 24 hours after dilution. However, a slight increase in particle size was observed in emulsions containing GLY. It was also found that the viscosity of emulsions increased with an increase S/O ratio as well as the concentration of the oil.