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.

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).

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.

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.

Stability of deslorelin injection.

The stability of deslorelin, a nonapeptide drug, in an injectable drug formulation was studied for 60 months. An HPLC analytical method was developed to quantify deslorelin and benzyl alcohol in deslorelin injection. The samples containing 500 micrograms/mL of deslorelin were stored at--10, 6, 25, 40 and 50 degrees C. No deslorelin degradation was observed in samples stored at 6 degrees C, but the loss of deslorelin in samples stored at 25, 40 and 50 degrees C was found to increase with temperature. The T90 of the product at 6 degrees C was estimated to be greater than 20 years from the short-term stability data of four lots stored at 25, 40 and 50 degrees C. The long-term stability results confirmed that there was no degradation of deslorelin stored at 6 degrees C for 5 years. Liquid secondary ionization mass spectrometry analysis of aged samples showed hydrolysis of deslorelin and the production of a hexapeptide (< Ser-Tyr-D-Trp-Leu-Arg-Pro-NH-Et) with a molecular weight of 847. A second product resulted from the oxidation of the tryptophan indoylyl ring. The oxyindoyle decapeptide structure had an ion m/z of 1298. The mass spectrum of pure deslorelin also is reported.

Expression and activity of hexokinase in the early mouse embryo.

The maximal activity and Michaelis constant, KM, of hexokinase have been measured in the peri-implantation mouse embryo using an ultramicrofluorescence technique. In addition, transcript detection of the predominant isoenzyme hexokinase I has been determined in single preimplantation mouse embryos at successive stages of development using reverse transcriptase-mediated cDNA amplification. Maximal hexokinase activity decreased dramatically peri-implantation, from 0.97 +/- 0.19 nmol/microgram protein/h at the blastocyst stage to 0.31 +/- 0.05 nmol/microgram protein/h on day 6.5. The KM remained relatively low and constant over this period (0.23-0.39 mM), indicating the absence of the hexokinase type IV isoenzyme. The pattern of hexokinase activity resembled that of glucose consumption suggesting a possible regulatory role for the enzyme during this period of development. Hexokinase I mRNA was detected in the oocyte and all preimplantation stages of development. The blastocyst polymerase chain reaction (PCR) product, when cloned and sequenced was found to be 98% homologous with mouse tumour hexokinase I. Taken together, these data suggest that the hexokinase gene is not under transcriptional control during early mouse embryo development but plays a significant role in the regulation of glucose consumption. A role for hexokinase in the phosphate-induced inhibition of early embryo development is also proposed.

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.

The regulation of actin polymerization in differentiating U937 cells correlates with increased membrane levels of the pertussis-toxin-sensitive G-protein Gi2.

Undifferentiated U937 cells appear to lack a capacity of increase cellular F-actin. However, electropermeabilized cells gain the ability to respond in this way to a guanine nucleotide analogue, guanosine 5'-[gamma-thio]trisphosphate (GTP[S]) after 1 h of treatment with dibutyryl cyclic AMP (db-cAMP). The results reported here show that the levels of membrane association of the G-protein Gi2 alpha increase with a time course identical with that of the GTP[S]-sensitivity of electropermeabilized cells. These results suggest that Gi2 alpha may be involved in the signal-transduction pathway leading to actin polymerization in db-cAMP-differentiated U937 cells.

An analysis of pollutant gas transport and absorption in pulmonary airways.

A mathematical model of ozone absorption, or for any soluble gas that has similar transport properties, is developed for a branching network of liquid-lined cylinders. In particular, we investigate specific flow regimes for finite length tubes where boundary layer phenomena and entrance effects exist in high Reynolds and Peclet (Pe) number airways. The smaller airways which have lower Reynolds and Peclet number flows are modelled by incorporating the detailed analysis found in [10] and modifying it for airways which have alveolated surfaces. We also consider a reacting gas and treat specific regimes where the reaction front is located at the air-liquid interface, within the liquid or at the liquid-tissue interface. Asymptotic methods are used in regions of the tracheobronchial tree where Pe much less than 1 and Pe much greater than 1. In addition, the fact that the radial transport parameter gamma much less than 1 for this toxin, and others such as nitrous oxides, is employed to simplify the analysis. The ozone concentrations, airway absorption and tissue dose are examined as a function of airway generation for several values of the governing parameters. The general result is a maximal dosing in airway generations 17 to 18 that is much larger (up to an order of magnitude) than the predictions of previous theories.

Dibutyryl cyclic AMP stimulation of a monocyte-like cell line, U937: a model for monocyte chemotaxis and Fc receptor-related functions.

Treatment of the U937 cell line with 1 mM dibutyryl cyclic AMP (Bt2cAMP) resulted in a reduction in cell size and inhibition of DNA synthesis, and morphologically the cells appeared similar to macrophages. Electron micrographs indicated an increase in intracellular apparatus, whilst histochemical studies revealed smaller, denser nuclei and a greater intensity of non-specific esterase staining. Ia-like antigens (HLA-DR and HLA-DC) and complement receptor CR1 were not detected on U937 cells by monoclonal antibodies, nor were they induced by Bt2cAMP. CR3 was present in small amounts on U937 cells, and stimulation with Bt2cAMP increased the expression of this molecule in the cytoplasm and on the cell surface. Leu M3, a monocyte-specific antibody, was weakly reactive on both unstimulated and stimulated cells, whereas transferrin receptors, present on 90% of U937 cells, were lost after 48-hr stimulation with Bt2cAMP. JW6 and NH6, two monoclonal antibodies raised in our laboratory and found to be against immature monocytic antigens, showed decreased expression on stimulation. Monomer IgG binding via Fc receptors decreased on stimulated cells, and a monoclonal antibody (32.2) specific for FcRI confirmed this to be due to a decrease in the number of high-affinity receptors, rather than a decrease in IgG-binding affinity. In contrast, expression of the low-affinity FcRII, monitored by monoclonal antibody IV3, increased dramatically after stimulation. Other functional changes included the production of superoxide anions and the induction of non-specific phagocytosis. Two dimensional gel analysis, of detergent soluble proteins from unstimulated and 48-hr stimulated U937 cells, showed many differences in protein expression. A detailed investigation of these changes will facilitate a better understanding of the molecular mechanisms involved in the differentiation of U937 cells.