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.

Sustained TL1A expression modulates effector and regulatory T-cell responses and drives intestinal goblet cell hyperplasia.

The tumor necrosis factor (TNF) superfamily protein TNF-like 1A (TL1A) is the ligand for death receptor 3 (DR3). TL1A is induced on activated dendritic cells (DCs) and its expression has been linked to human inflammatory bowel disease. To address how TL1A might influence intestinal inflammation, we generated transgenic mice that constitutively express TL1A on DCs. TL1A transgenic mice developed striking goblet cell hyperplasia in the ileum that was associated with elevated interleukin (IL)-13 levels in the small intestine. IL-13- and IL-17-producing small intestinal lamina propria T cells were increased in TL1A transgenic mice. TL1A also enhanced regulatory T (Treg) cell turnover in vivo and directly stimulated Treg cell proliferation in vitro. The presence of TL1A attenuated the ability of Treg cells to suppress conventional T cells, an effect that required DR3 signaling in either conventional T cells or Treg cells. Our findings identify mechanisms by which chronic DR3 signaling could promote pathogenesis in inflammatory bowel disease.

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.

Ocular complications of pregnancy.

Pregnancy is often associated with ocular changes, most often transient in nature, though occasionally permanent. It can be associated with development of new conditions, or can exacerbate pre-existing conditions. The ocular effects of pregnancy may be divided into physiologic changes, pathologic conditions or modifications of pre-existing conditions. Pathologic conditions include entities such as pre-eclampsia and eclampsia, along with conditions that are seen with increased frequency during pregnancy such as central serous retinopathy. The most significant modified pre-existing condition is diabetes mellitus. The various effects of pregnancy on the eye will be reviewed in this article.

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.

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.

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.

Assembly of tight junctions during early vertebrate development.

Tight junction formation during development is critical for embryonic patterning and organization. We consider mechanisms of junction biogenesis in cleaving mouse and Xenopus eggs. Junction assembly follows the establishment of cell polarity at 8-cell (mouse) or 2-cell (Xenopus) stages, characterized by sequential membrane delivery of constituents, coordinated by embryonic (mouse) or maternal (Xenopus) expression programmes. Cadherin adhesion is permissive for tight junction construction only in the mouse. Occludin post-translational modification and membrane delivery, mediated by delayed ZO-1 alpha(+)isoform expression in the mouse, provides a mechanism for completion of tight junction biogenesis and sealing, regulating the timing of blastocoel cavitation.

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.

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.

Post-translational control of occludin membrane assembly in mouse trophectoderm: a mechanism to regulate timing of tight junction biogenesis and blastocyst formation.

The mouse blastocyst forms during the 32-cell stage with the emergence of the blastocoelic cavity. This developmental transition is dependent upon the differentiation and transport function of the trophectoderm epithelium which forms the wall of the blastocyst and exhibits functional intercellular tight junctions (TJs) to maintain epithelial integrity during blastocoele expansion. To investigate mechanisms regulating the timing of blastocyst formation, we have examined the dynamics of expression of occludin, an integral membrane protein of the TJ. Confocal microscopy of intact embryos and synchronised cell clusters revealed that occludin first assembles at the apicolateral membrane contact site between nascent trophectoderm cells usually during the early 32-cell stage, just prior to the time of blastocoele cavitation. This is a late event in the assembly of TJ-associated proteins within trophectoderm which, from our previous data, spans from 8- to 32-cell stages. Occludin membrane assembly is dependent upon prior E-cadherin-mediated cell-cell adhesion and is sensitive to brefeldin A, an inhibitor of Golgi-to-membrane transport. Occludin is delivered to the TJ site in association with the TJ plaque protein, ZO-1(&agr;)+, which we have shown previously is newly transcribed and translated during late cleavage. Immediately after assembly and before cavitation, occludin localised at the TJ site switches from a Triton X-100-soluble to -insoluble form indicative of actin cytoskeletal and/or membrane anchorage. Occludin mRNA and protein are detectable throughout cleavage by RT-PCR and immunoblotting, respectively, indicating that timing of membrane assembly is not controlled by expression alone. Rather, we have identified changes in the pattern of different occludin forms expressed during cleavage which, using phosphatase treatment of embryo lysates, include post-translational modifications. We propose that the phosphorylation of one form of occludin (band 2, 65-67 kDa) during late cleavage, which leads to its exclusive conversion from a Triton X-100-soluble to -insoluble pool, may regulate occludin association with ZO-1(&agr;)+ and membrane assembly, and thereby act to control completion of TJ biogenesis and the timing of blastocyst formation.

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.

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).

In space, the past can be recast but not the present.

We address the relationship between perception and spatial, working memory. Specifically, we argue that perceptual experience following the creation of a representation of target location affects it in a systematic way. We designed a motor task in which observers had to point to the initial or final position of a horizontally drifting target embedded in a vertically drifting background. The target was perceived as having an illusory motion component in a direction opposite that of the inducer dots [Duncker, 1938, Source Book of Gestalt Psychology (London: Kegan Paul, Trench, Trubner and Co)]. For both positions, there was an identical time delay before the observer could respond. Nonetheless, estimates of the initial target position were significantly biased by the illusion in a direction opposite the perceived target motion, and both bias and variability were significantly greater than those of the target's final position. In prior studies on positional accuracy with induced displacement, a delay before a pointing response led to an unbiased position estimate obtained without delay to become biased, leading investigators to argue for a long-lasting, inaccurate cognitive system that overrules an accurate, nonetheless transient, motor one (Bridgeman et al, 1997, Perceptual Psychology 59 456-469). Since the same motor task with identical delay on either position yielded different outcomes, a hypothesis based on distinct motor and cognitive representations of visual space is untenable here. Instead, we argue that an online representation of the target's original position is updated in an ongoing fashion in order to reconcile the perceived illusion with the veridically perceived present (current target location).

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.

A novel self emulsifying parenteral drug delivery system.

The application of three polyhydroxy alcohols for improving parenteral emulsion formulations was investigated. A mixture of lecithin, as the primary emulsifier, and Span 20 as the secondary emulsifier, was used as the emulsifier system. The polyhydroxy alcohols selected were glycerol, propylene glycol and sorbitol. Soybean oil-in-water emulsions were prepared with the addition of increasing concentrations of each polyhydroxy alcohol. It was found that anhydrous mixtures of oil, surfactants and 30% or higher concentration of glycerol formed self emulsifying isotropic liquids, suitable for preparing Parenteral Self Emulsifying Drug Delivery Systems (PSEDDS). Spontaneous emulsification to submicron particle size of 0.4 micron occurred when these isotropic liquids were gently mixed with water. A PSEDDS formulation, containing 0.5% lidocaine, as the model drug showed similar spontaneous emulsification with particle size of 0.39 micron. Formulations containing propylene glycol, or sorbitol or lower concentrations of glycerol did not form self emulsifying mixtures. There were substantial differences in the particle size reduction pattern with each polyhydroxy alcohol. Glycerol was most effective, with minimum particle size obtained at 30% concentration. Addition of propylene glycol resulted in minimum particle size at 60% concentration. But there was increase in particle size at higher concentrations. Sorbitol was not very effective in reducing particle size. Alteration of the surfactant phase distribution at the interface was found to be the primary effect of polyhydroxy alcohols.

Effect of lyophilization on parenteral emulsions.

The feasibility of preparing lyophilized anhydrous products, for reconstitution in to emulsion dosage forms was investigated. Stable soybean o/w emulsions were prepared using a mixture of lecithin and Span 20 as the emulsifiers. Two series of emulsions were prepared for this study, each containing a polyhydroxy alcohol as a consurfactant for particle size reduction. Increasing concentrations of glycerol (10-30% w/w) were added to one group of emulsions and propylene glycol (20-60% w/w) was added to the second group of emulsions. All formulations were found to have good particle size stability. The emulsion formulation containing 30% glycerol could be successfully lyophilized into an anhydrous product. Reconstitution of this lyophilized product resulted in an emulsion essentially similar to the original emulsion prior to lyophilization. This is because the mixture of the oil phase and 30% w/w glycerol formed a self-emulsifying system. All other emulsion formulations were not suitable for lyophilization. These formulations cracked during lyophilization, separating into an upper oil layer and a lower layer of the continuous phase. The formation of an upper oil layer prevented complete drying of these emulsions. The particle size of these lyophilized emulsions, when reconstituted with the external phase was greater than the emulsion particle size prior to lyophilization. But the change in particle size was less with increasing concentrations of polyhydroxy alcohols. These results indicate that emulsions can be lyophilized to prepare a product suitable for reconstitution to a parenteral emulsion dosage form provided the formulation is designed to withstand temperature and phase changes during the lyophilization process.

Sequence analysis of V(4-34)-encoded antibodies from single B cells of two patients with systemic lupus erythematosus (SLE).

SLE is an autoimmune disease characterized by the presence of autoantibodies against double-stranded (ds)DNA. A large proportion (approx. 40%) of patients with lupus also have increased levels of serum immunoglobulin encoded by the V(4-34) heavy chain gene, which often fluctuate with disease activity, and this gene is utilized by a subset of anti-dsDNA antibodies. In order to probe the nature of the V(4-34)-encoded immunoglobulin, B cells were isolated from the blood of two patients with active disease, using the 9G4 MoAb specific for the immunoglobulin gene product. Following cell picking, single-cell polymerase chain reaction (PCR) amplification of cDNA was used to investigate both V(H) and V(L) genes. Sequences were obtained from B cells synthesizing IgM (n = 10), IgG (n = 4) and IgA (n = 1). For V(H), all were derived from V(4-34) as expected, and the isotype-switched sequences and 2/6 IgM sequences were somatically mutated. In contrast, V(L) (12 kappa and 3 lambda) showed a low level of mutation, possibly indicating secondary rearrangements. The three most highly mutated V(H) sequences were associated with unmutated V(L) sequences. Analysis of the distribution of mutations revealed only minor clustering in complementarity-determining regions (CDRs) characteristic of antigen selection. The CDR3 lengths of V(H) ranged from five to 19 amino acids, and in 3/15 there was evidence of an excess of positively charged amino acids, compared with the normal expressed repertoire. Basic amino acids were also found at the V(L)-J(L) junctions in 4/15. These findings provide insight into the V(4-34)-V(L) gene combinations used by B cells in patients with SLE which might have clinical relevance.

Temporal modulation of spatial borders in rat barrel cortex.

We examined the effects of varying vibrissa stimulation frequency on intrinsic signal and neuronal responses in rat barrel cortex. Optical imaging of intrinsic signals demonstrated that the region of cortex activated by deflection of a single vibrissa at 1 Hz is more diffuse and more widespread than the territory activated at 5 or 10 Hz. With the use of two different paradigms, constant time of stimulation and constant number of vibrissa deflections, we showed that the optically imaged spread of activity is more discrete at higher stimulation frequencies. We combined optical imaging with multiple electrode recording and confirmed that the neuronal response to individual vibrissa stimulation at the optically imaged center of activity is greater than the response away from the imaged center. Consistent with the imaging data, these recordings also showed no response to a second vibrissa deflection at 5 Hz at a peripheral recording site, though there was a significant response to a second vibrissa deflection at 1 Hz at the same peripheral site. These findings demonstrate that vibrissa stimulation at higher frequencies leads to more focused physiological responses in cortex. Thus the spread of activation in rat barrel cortex is modulated in a dynamic fashion by the frequency of vibrissa stimulation.

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.