Health information quality on the internet in gynecological oncology: a multilingual evaluation.

BACKGROUND: Oncological internet information quality is considered variable, but no comprehensive analysis of gynecological malig- nancies exists. The present authors' objectives were to compare the quality of common malignancy websites and to assess for language or disease differences; and secondly, to perform a quality comparison between medical and layperson terminology. MATERIALS AND METHODS: World Health Organization (WHO) Health on the Net (HON) principles may be applied to websites using an automated toolbar function. Using a search engine (www.Google.com) 8,400 websites were assessed using keywords 'endometrial, 'uterine', 'cervical', 'ovarian', 'vaginal', 'vulvar', plus 'cancer', in English, French, German, and Spanish; repeated for alternate terms e.g. 'cervix', 'womb'. RESULTS: Searches for "vaginal' 'uterine', 'cervical', and 'endometrial' each returned millions of websites. The total percentage of all assessed HON-accredited sites was notably low across all search terms (median 15%; range 3-19%). Significant differences by malignancy type (p < 0.0001), language (p < 0.0001), and tertiles (thirds) of the first 150 websites returned (p < 0.0001). French language had most accredited websites. Using alternate terms demonstrated significant differences (p < 0.001) in accredited websites for most gynecological cancers. CONCLUSIONS: Internet data on gynecological malignancies is overwhelming. Further, a lack of validation of the majority of gynecological oncologic sites should be appreciated with discrepancies in quality and number of websites across diseases, languages, and also between medical and layperson terms. Physicians should encourage and more importantly their professional bodies should participate in the development of informative, ethical, and reliable health websites on the internet and direct patients to them.

Lignocaine plasma levels following topical gel application in laparoscopic and hysteroscopic procedures.

The study aim was to determine plasma lignocaine concentrations resulting from topical application of a newly formulated, sterile two-pack lignocaine gel in laparoscopic and hysteroscopic procedures. This was an open label single-centre study in which six female patients underwent laparoscopy and six underwent hysteroscopy. One venous blood sample was extracted pre-gel application, followed by 10 samples over a 24 hour period following application. Samples were centrifuged, stored at -20°C and subsequently analysed for lignocaine and its metabolite, monoethyl-glycinexylidide. Application of gel in doses between 2.7 and 5.8 mg/kg of lignocaine resulted in a maximum plasma concentration in any patient of 1520 ng/ml lignocaine and 240 ng/ml monoethyl-glycinexylidide. These maximum concentrations were recorded in a patient undergoing a laparoscopic procedure and patients undergoing hysteroscopic procedures all recorded lower maximum concentrations compared with patients undergoing laparoscopy; the maximum observed concentrations in a patient having a hysteroscopy were 420 ng/ml lignocaine and 56 ng/ml of monoethyl-glycinexylidide.A new sterile two-pack topical lignocaine gel, applied at the end of laparoscopic and hysteroscopic procedures in doses up to 5.84 mg/kg, resulted in plasma lignocaine levels below those known to have the potential to cause central nervous system toxicity.

An analysis of lymphocyte phenotype after steroid avoidance with either alemtuzumab or basiliximab induction in renal transplantation.

Several studies have analyzed the phenotype of repopulated T-lymphocytes following alemtuzumab induction; however there has been less scrutiny of the reconstituted B-cell compartment. In the context of a randomized controlled trial (RCT) comparing alemtuzumab induction with tacrolimus monotherapy against basiliximab induction with tacrolimus and mycophenolate mofetil (MMF) therapy in renal transplantation, we analyzed the peripheral B- and T-lymphocyte phenotypes of patients at a mean of 25 +/- 2 months after transplantation. We examined the relationship between peripheral lymphocyte phenotype and graft function. Patients who received alemtuzumab had significantly higher numbers of B cells including naïve, transitional and regulatory subsets. In contrast, the CD4(+) T-cell compartment was dominated by a memory cell phenotype. Following either basiliximab or alemtuzumab induction patients with lower numbers of B cells or B subsets had significantly worse graft function. For alemtuzumab there was also a correlation between these subsets the stability of graft function and the presence of HLA-specific antibodies. These results demonstrate that a significant expansion of regulatory type B cells is associated with superior graft function and that this pattern is more common after alemtuzumab induction. This phenomenon requires further prospective study to see whether this phenotype could be used to customize immunotherapy.

Specific glycosaminoglycans promote unseeded amyloid formation from beta2-microglobulin under physiological conditions.

Dialysis-related amyloidosis (DRA) is a complication of hemodialysis where beta2-microglobulin (beta2m) forms plaques mainly in cartilaginous tissues. The tissue-specific deposition, along with a known intransigence of pure beta2m to form fibrils in vitro at neutral pH in the absence of preformed fibrillar seeds, suggests a role for factors within cartilage in enhancing amyloid formation from this protein. To identify these factors, we determined the ability of a derivative lacking the N-terminal six amino acids found in ex vivo beta2m amyloid deposits to form amyloid fibrils at pH 7.4 in the absence of fibrillar seeds. We show that the addition of the glycosaminoglycans (GAGs) chrondroitin-4 or 6-sulfate to fibril growth assays results in the spontaneous generation of amyloid-like fibrils. By contrast, no fibrils are observed over the same time course in the presence of hyaluronic acid, a nonsulfated GAG that is abundant in cartilaginous joints. Based on the observation that hyaluronic acid has no effect on fibril stability, while chrondroitin-6-sulfate decreases the rate of fibril disassembly, we propose that the latter GAG enhances amyloid formation by stabilizing the rare fibrils that form spontaneously. This leads to the accumulation of beta2m in fibrillar deposits. Our data rationalize the joint-specific deposition of beta2m amyloid in DRA, suggesting mechanisms by which amyloid formation may be promoted.

Effects of oxygen tension on the establishment and lactate dehydrogenase activity of murine embryonic stem cells.

The lack of a standardized culture environment for establishment of embryonic stem cell lines has hindered the orchestrated differentiation of cells and the application of this technology. Oxygen concentration has a profound effect on proliferation and differentiation of many cell types. This study tested the hypothesis that establishment dynamics, lactate dehydrogenase (LDH) isoforms, and mRNA expression patterns would be affected by the oxygen tension in the culture environment. Recovered (day 4) murine blastocysts were cultured in a gas environment of 6% CO(2) and either 20% or 5% O(2) (balance supplemented with N(2)). More (p < 0.05) blastocysts produced outgrowths in the low (79.3 +/- 0.1%) compared to the high (57.1 +/- 0.1%) O(2) groups, and more (p < 0.05) colonies in the low O(2) group (14/15; 93.3 +/- 0.1%) stained positive for alkaline phosphatase relative to the high O(2) group (9/15; 60.6 +/- 0.1%). Oxygen treatment had no effect on the activity of the oxioreductase lactate dehydrogenase. Interestingly, the stem cell lines in both treatments displayed multiple isoforms (III, IV, and V) of LDH, whereas the outgrowths displayed isoforms I and V. In contrast, two-cell embryos and blastocysts displayed only isoform I, and fibroblasts displayed isoforms IV and V. There were no treatment differences in mRNA expression of LDHalpha in the outgrowths, or established stem cells. LDH transition from the heart (I) to the muscle (V) isoform indicated an increase in glycolytic activity, consistent with the peri-hatching/implantation time period. Reduced O(2) environment had significant positive effects on the establishment and maintenance of murine stem cells, supporting the hypothesis, whereas the LDH isozyme transition was consistent among treatments.

Evolution of IESs and scrambling in the actin I gene in hypotrichous ciliates.

Germ-line (micronuclear) genes in hypotrichous ciliates are interrupted by numerous, short, noncoding, AT-rich segments called internal eliminated segments, or IESs. IESs divide a gene into macronuclear destined segments, or MDSs. IESs are excised from micronuclear genes, and the MDSs are spliced when a micronuclear genome is processed into a macronuclear genome after cell mating. In the micronuclear version of the actin I gene intramolecular recombination between IESs during evolution has put MDSs into a scrambled disorder in some but not all hypotrichs. Studies using rDNA sequences to define phylogenetic relationships among eight hypotrichs suggests that evolution of the micronuclear actin I gene proceeds by successive addition of IESs in earlier diverging species, without MDS scrambling. Continued addition of IESs and recombination among IESs in later diverging species produced actin I genes with scrambled MDSs. Subsequent to MDS scrambling, additional IESs were inserted into the more recently evolved species. Thus, IES insertions and gene scrambling occur in a progressive manner during species evolution to produce micronuclear actin I genes of increasing structural complexity.

Human oesophagostomiasis: a histomorphometric study of 13 new cases in northern Ghana.

Oesophagostomiasis is an infrequently described and recognised parasitic infection in humans, caused by Oesophagostomum bifurcum. Although the disease is most often found in the northern part of Togo and the neighbouring part of Ghana, sporadic cases have been described in other parts of Africa and in Asia and South America: Uganda, Ivory Coast, Sudan, Kenya, Ethiopia, Indonesia, Malaysia and Brazil. Infection probably occurs by way of the ingestion of L3 larvae. These larvae penetrate the intestinal wall, especially that of the colon. Some of these larvae develop into young adult worms and return to the bowel lumen. Other larvae, however, develop into immature worms, which fail to settle in the lumen, forming abscesses in the bowel wall and causing pathology. In the literature 105 human cases have been described, many originating in the northern regions of Ghana and Togo. The present study was performed to evaluate 13 new cases originating in the northern part of Ghana (7 female and 6 male patients, aged between 2 and 60 years). Histopathologically, the patients could be divided into two groups: the first group showed multinodular disease, while patients in the second group presented with a single, nodular mass. In the first group, abscesses were seen throughout the colonic wall. The mean size of the cavities was 4.3+/-0.7 mm. There was no relation between the size and the localisation in the colonic wall. Abscesses were significantly larger in male patients than in female patients. There was no correlation with age. In the second group, histopathological examination showed a cyst of variable wall thickness with very limited inflammation. These cysts represented older lesions, often encapsulated in the mesentery. In conclusion, in this study we present 13 new cases of human oesophagostomiasis. The abscess formation was found to be organ specific, independent of age, and gender-related, producing a more intense tissue reaction in male patients.

Distinct functions and cooperative interaction of the subunits of the transporter associated with antigen processing (TAP).

The ATP-binding cassette (ABC) transporter TAP translocates peptides from the cytosol to awaiting MHC class I molecules in the endoplasmic reticulum. TAP is made up of the TAP1 and TAP2 polypeptides, which each possess a nucleotide binding domain (NBD). However, the role of ATP in peptide binding and translocation is poorly understood. We present biochemical and functional evidence that the NBDs of TAP1 and TAP2 are non-equivalent. Photolabeling experiments with 8-azido-ATP demonstrate a cooperative interaction between the two NBDs that can be stimulated by peptide. The substitution of key lysine residues in the Walker A motifs of TAP1 and TAP2 suggests that TAP1-mediated ATP hydrolysis is not essential for peptide translocation but that TAP2-mediated ATP hydrolysis is critical, not only for translocation, but for peptide binding.

Induction of tyrosinase gene transcription in human iris organ cultures exposed to latanoprost.

OBJECTIVE: Topical administration of latanoprost sometimes induces gradual iris darkening. The present study was undertaken to determine if latanoprost can increase transcription of the gene for tyrosinase, an important enzyme in the biosynthesis of melanin. Results from brown, hazel, and blue irides were compared. METHODS: Iris tissue was isolated from 30 pairs of postmortem human donor eyes, and 2 iris segments from each eye were incubated in tissue culture medium supplemented with 200nM latanoprost acid or vehicle for 7 days. Tyrosinase messenger RNA (mRNA) was determined using real-time polymerase chain reaction analysis (TaqMan quantitative polymerase chain reaction). Results for tyrosinase mRNA were normalized according to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA in each sample. RESULTS: Tyrosinase mRNA expression was similar in blue and hazel irides, and ranged from 0.7-fold to 12.6-fold greater than GAPDH expression. In contrast, control brown iris culture tyrosinase expression ranged from 6.4-fold to 265-fold greater than GAPDH expression. Induction of tyrosinase mRNA by latanoprost was below threshold in all the blue iris cultures (n = 8 pairs), present in 1 of 9 hazel iris cultures, and present in 5 of 13 brown iris cultures. Mean induction in the responding hazel iris cultures was 1.40-fold. Mean induction among the responding brown iris cultures was 2.97-fold. CONCLUSIONS: These observations support the view that iris darkening associated with latanoprost treatment reflects induction of tyrosinase expression. Moreover, they suggest that the probability that latanoprost will increase tyrosinase expression is directly related to the magnitude of tyrosinase expression before treatments are initiated. CLINICAL RELEVANCE: The variability of iris darkening with latanoprost may reflect natural variation in the basal transcription of tyrosinase.

Interaction of cigarette smoke and house dust mite allergens on inflammatory mediator release from primary cultures of human bronchial epithelial cells.

Several studies have shown that exposure to cigarette smoke and/or house dust mite (HDM) can lead to increased airway inflammation in susceptible individuals. The underlying mechanisms, however, are not defined. To investigate the interaction between cigarette smoke and HDM allergen on mediator release from primary cultures of human bronchial epithelial cells. Confluent human bronchial epithelial cell cultures were exposed to cigarette smoke in the absence or presence of HDM allergen and investigated for the release of IL-8, IL-1beta, and sICAM-1. Damage to the epithelial cells themselves was assessed by release of 51Cr. On separate occasions, we investigated the effect of PTL11028, a highly potent and selective Der p1 inhibitor, on HDM allergen-induced release of IL-8, following activation of HDM allergen by incubation with cysteine. The effect of cigarette smoke exposure on the stability of these released mediators in prepared solutions in the absence/presence of reduced glutathione was also studied. Both HDM allergens and short-term (20 min) cigarette smoke exposure led to a significantly increased release of IL-8, IL-1beta and sICAM-1 from the epithelial cell cultures. Longer exposure (1-6 h) to cigarette smoke led to a dramatic decrease in the amount of these mediators detected in the culture medium. Whilst incubation of epithelial cultures with HDM allergen did not cause any significant change in the release of 51Cr from pre-loaded cells, cigarette smoke on its own led to a marked, exposure and incubation-time dependent increase in the release of 51Cr. Incubation with HDM allergen led to a significant, dose and time-dependent increase in the release of IL-8, which was further enhanced when the allergen extract was pre-activated with cysteine. This effect was completely abrogated by PTL11028, a novel Der p1 inhibitor. Prepared solutions of various concentrations of IL-8, IL-1beta and sICAM-1 exposed to cigarette smoke demonstrated a dramatic exposure time-dependent decrease in the detectable amount of these mediators, an effect which was abrogated by GSH. HDM-induced airway inflammation may include Der p-mediated release of inflammatory mediators from epithelial cells. Additionally, short-term cigarette smoke exposure may induce airway inflammation by release of inflammatory mediators from these cells, an effect which may be potentiated by Der p allergens. Longer term cigarette smoke exposure may cause damage to epithelial cells and changes in the structure of inflammatory mediators.

The human cytomegalovirus gene product US6 inhibits ATP binding by TAP.

Human cytomegalovirus (HCMV) encodes several genes that disrupt the major histocompatibility complex (MHC) class I antigen presentation pathway. We recently described the HCMV-encoded US6 gene product, a 23 kDa endoplasmic reticulum (ER)-resident type I integral membrane protein that binds to the transporter associated with antigen processing (TAP), inhibits peptide translocation and prevents MHC class I assembly. The functional consequence of this inhibition is to prevent the cell surface expression of class I bound viral peptides and their recognition by HCMV-specific cytotoxic T cells. Here we describe a novel mechanism of action for US6. We demonstrate that US6 inhibits the binding of ATP by TAP1. This is a conformational effect, as the ER lumenal domain of US6 is sufficient to inhibit ATP binding by the cytosolic nucleotide binding domain of TAP1. US6 also stabilizes TAP at 37 degrees C and prevents conformational rearrangements induced by peptide binding. Our findings suggest that the association of US6 with TAP stabilizes a conformation in TAP1 that prevents ATP binding and subsequent peptide translocation.

Antigen presentation: peptides and proteins scramble for the exit.

The fate of peptides that fail to bind to major histocompatibility complex class I molecules in the endoplasmic reticulum (ER)has remained unclear. A recent study has revealed that these peptides exit the ER via the Sec61 channel and compete for this pathway with misfolded proteins.

Clinical epidemiology and classification of human oesophagostomiasis.

The intestinal helminth Oesophagostomum bifurcum is highly and focally endemic in northern Ghana and Togo, and its juveniles produce a nodular inflammatory response as they develop in the intestinal wall. This pathology can produce clinical symptoms. We report on 156 cases of oesophagostomiasis presenting in 1996-98 to Nalerigu hospital in northern Ghana. The disease accounted for 0.2% of the out-patient department new presentations (about 1 patient per week), and 1% (16) of the major acute surgical cases. Children aged 5-9 years were most commonly affected. Multinodular disease (13% of the cases) results from hundreds of pea-sized nodules within the colon wall and other intra-abdominal structures, and presents with general abdominal pain, persistent diarrhoea and weight loss. Dapaong tumour (87%) presents as an abdominal inflammatory mass often associated with fever. The 3-6-cm tumour is painful, well-delineated, smooth, spherical, 'wooden', periumbilical, and adhered to the abdominal wall. Cases most commonly presented during the late rains and early dry season. Diagnosis by ultrasound has reduced the need for exploratory surgery, and the ability to sonographically evaluate conservative treatment with albendazole has curtailed management by colectomy or incision and drainage.

Functional effects of the inhibition of the cysteine protease activity of the major house dust mite allergen Der p 1 by a novel peptide-based inhibitor.

BACKGROUND: The house dust mite (HDM) Dermatophagoides pteronyssinus is an important source of allergens, which can cause allergic conditions. The cysteine protease activity of Der p 1 may enhance the potency of this major mite allergen through cleavage of CD23 and CD25 from the surface of immune cells, IgE independent mast cell activation, increases in epithelial cell permeability and inactivation of an endogenous serine protease inhibitor. Inhibition of the enzymatic activity of Der p 1 may therefore be of therapeutic benefit. OBJECTIVE: To examine the activity of PTL11028, a newly developed Der p 1 inhibitor, in a range of assays that directly or indirectly measure Der p 1 protease activity and to compare its activity to endogenous cysteine protease inhibitors. METHODS: The proteolytic activities of purified Der p 1 or HDM extract and inhibitory properties of PTL11028 were examined through cleavage of an artificial peptidyl substrate, cleavage of CD23 from human B cells and permeability studies on primary human bronchial epithelial cells. RESULTS: PTL11028 is a highly potent and specific Der p 1 inhibitor, being effective against both purified protease and Der p 1 within HDM extract. PTL11028 can completely inhibit Der p 1-mediated CD23 cleavage from human B cells and also reduces HDM-induced human bronchial epithelial cell permeability by 50%. Der p 1 is potently inhibited by cystatin A and to a lesser extent by cystatins C and E/M. CONCLUSION: PTL11028 is a highly potent and selective irreversible inhibitor of the cysteine protease activity of Der p 1, an activity that may be modulated in vivo by some human cystatins. PTL11028 prevents the Der p 1-mediated cleavage of CD23 from human B cells and significantly reduces HDM-induced permeabilization of the epithelial barrier. PTL11028 is an important tool to examine the biological effects of Der p 1 in a range of in vitro and in vivo model systems.

A functional role for interleukin (IL)-4-driven cyclic amp accumulation in human b lymphocytes.

Interleukin-4 (IL-4) regulates the expression of the 55-kDa alpha-subunit (CD25) of the IL-2 receptor complex in human B lymphocytes. This report suggests that the cAMP/protein kinase A (PKA) component of the IL-4 receptor signalling programme in human tonsillar B cells has a functionally important role in regulating expression of the CD25 gene by attenuating activity of a protein binding to a potent negative regulatory element (NRE) in the CD25 promoter; this effect can be mimicked by agents that elevate cAMP and blocked by inhibitors of PKA but not protein kinase C (PKC). In a B-cell line that fails to elevate cAMP, attenuate NRE-binding protein (NRE-BP) activity or express CD25 following IL-4 treatment, stimulation of cAMP accumulation by forskolin facilitates IL-4-mediated induction of both the endogenous gene and an exogenous reporter gene under the control of a minimal promoter/enhancer fragment of the CD25 gene.

The structures of the H(C) fragment of tetanus toxin with carbohydrate subunit complexes provide insight into ganglioside binding.

The entry of tetanus neurotoxin into neuronal cells proceeds through the initial binding of the toxin to gangliosides on the cell surface. The carboxyl-terminal fragment of the heavy chain of tetanus neurotoxin contains the ganglioside-binding site, which has not yet been fully characterized. The crystal structures of native H(C) and of H(C) soaked with carbohydrates reveal a number of binding sites and provide insight into the possible mode of ganglioside binding.

Di-leucine signals mediate targeting of tyrosinase and synaptotagmin to synaptic-like microvesicles within PC12 cells.

One pathway in forming synaptic-like microvesicles (SLMV) involves direct budding from the plasma membrane, requires adaptor protein 2 (AP2) and is brefeldin A (BFA) resistant. A second route leads from the plasma membrane to an endosomal intermediate from which SLMV bud in a BFA-sensitive, AP3-dependent manner. Because AP3 has been shown to bind to a di-leucine targeting signal in vitro, we have investigated whether this major class of targeting signals is capable of directing protein traffic to SLMV in vivo. We have found that a di-leucine signal within the cytoplasmic tail of human tyrosinase is responsible for the majority of the targeting of HRP-tyrosinase chimeras to SLMV in PC12 cells. Furthermore, we have discovered that a Met-Leu di-hydrophobic motif within the extreme C terminus of synaptotagmin I supports 20% of the SLMV targeting of a CD4-synaptotagmin chimera. All of the traffic to the SLMV mediated by either di-Leu or Met-Leu is BFA sensitive, strongly suggesting a role for AP3 and possibly for an endosomal intermediate in this process. The differential reduction in SLMV targeting for HRP-tyrosinase and CD4-synaptotagmin chimeras by di-alanine substitutions or BFA treatment implies that different proteins use the two routes to the SLMV to differing extents.

A complex web of signal-dependent trafficking underlies the triorganellar distribution of P-selectin in neuroendocrine PC12 cells.

By analyzing the trafficking of HRP-P-selectin chimeras in which the lumenal domain of P-selectin was replaced with horseradish peroxidase, we determined the sequences needed for targeting to synaptic-like microvesicles (SLMV), dense core granules (DCG), and lysosomes in neuroendocrine PC12 cells. Within the cytoplasmic domain of P-selectin, Tyr777 is needed for the appearance of P-selectin in immature and mature DCG, as well as for targeting to SLMV. The latter destination also requires additional sequences (Leu768 and 786DPSP789) which are responsible for movement through endosomes en route to the SLMV. Leu768 also mediates transfer from early transferrin (Trn)-positive endosomes to the lysosomes; i.e., operates as a lysosomal targeting signal. Furthermore, SLMV targeting of HRP-P-selectin chimeras, but not the endogenous SLMV protein synaptophysin/p38, previously shown to be delivered to SLMV directly from the plasma membrane, is a Brefeldin A-sensitive process. Together, these data are consistent with a model of SLMV biogenesis which involves an endosomal intermediate in PC12 cells. In addition, we have discovered that impairment of SLMV or DCG targeting results in a concomitant increase in lysosomal delivery, illustrating the entwined relationships between routes leading to regulated secretory organelles (RSO) and to lysosomes.

Cigarette smoke potentiates house dust mite allergen-induced increase in the permeability of human bronchial epithelial cells in vitro.

Although studies have suggested that exposure to cigarette smoke (CS) may be associated with the development of atopy, the mechanisms underlying this are not clearly understood. It has been suggested that CS impairs the barrier function of the airway epithelium, leading to increased access of allergens such as those of the house dust mite (HDM) Dermatophagoides pteronyssinus (Der p) to antigen-presenting cells, with subsequent allergic sensitization. In order to test this hypothesis, we established primary explant cultures of human bronchial epithelial cells (HBEC) in cell culture inserts, and exposed these for 20 min, 1 h, 3 h, and 6 h to CS or air in the absence or presence of 300 ng/ml Der p, and then further incubated the cultures over a period of 24 h. The HBEC cultures were assessed for changes in permeability as measured by changes in: (1) electrical resistance (ER); and (2) passage of 14C-labeled bovine serum albumin (14C-BSA) and Der p allergens across the HBEC cultures. We also assessed the effects of protease inhibitors and the antioxidant glutathione (GSH) in this experimental system. Damage to HBEC cultures was assessed by the release of [51Cr]sodium chromate from prelabeled cells, and by release of lactate dehydrogenase (LDH). Twenty minutes of exposure to CS as compared with exposure to air did not significantly alter either the ER or passage of 14C-BSA across the HBEC cultures. In contrast, incubation with Der p led to a significant increase in the permeability of HBEC cultures, an effect that was enhanced by exposure to CS but was abrogated by the specific protease inhibitors and GSH. Passage of Der p was also increased by exposure to CS. Exposure of HBEC cultures to CS led to a significant release of 51Cr and LDH from these cells as compared with cells exposed to air. This effect was augmented further when HBEC cultures were incubated with Der p. Exposure of HBEC cultures for 1 h, 3 h, and 6 h to CS led to a markedly significant dose- and time-dependent increase in the permeability of these cells. These results suggest that exposure to CS significantly enhances Der p-induced decreases in electrical resistance and the increased passage across HBEC cultures of 14C-BSA and of the Der p allergen itself.

A balance of opposing signals within the cytoplasmic tail controls the lysosomal targeting of P-selectin.

The 35-amino acid cytoplasmic tail of the adhesion receptor P-selectin is subdivided into stop transfer, C1 and C2 domains. It contains structural signals needed for targeting this protein to specialized secretory organelles and to lysosomes. Recently, using site-directed mutagenesis of horseradish peroxidase-P-selectin chimeras, we have uncovered a novel sequence within the C1 domain, KCPL, that mediates sorting from early, transferrin-positive endosomes to lysosomes and therefore operates as a positive lysosomal targeting signal (Blagoveshchenskaya, A. D., Norcott, J. P. , and Cutler, D. F. (1998) J. Biol. Chem. 273, 2729-2737). In the current study, we examined lysosomal targeting by both subcellular fractionation and an intracellular proteolysis assay and found that a balance of positive and negative signals is required for proper lysosomal sorting of P-selectin. First, we have found that within the sequence KCPL, Cys-766 plays a major role along with Pro-767, whereas Lys-765 and Leu-768 make no contribution to promoting lysosomal targeting. In addition, horseradish peroxidase-P-selectin chimeras were capable of acylation in vivo with [3H]palmitic acid at Cys-766, since no labeling of a chimera in which Cys-766 was replaced with Ala was detected. Second, analysis of mutations within the C2 domain revealed that substitution of two sequences, YGVF and DPSP, causes an increase in both lysosomal targeting and intracellular proteolysis suggesting the presence of lysosomal avoidance signals. The inhibition or promotion of lysosomal targeting resulted from alterations in endosomal sorting since internalization was not changed in parallel with lysosomal delivery. Analysis of the double mutants KCPL/YGVF or KCPL/DPSP revealed that although the positive lysosomal targeting signal operates in the early/sorting transferrin-positive endosomes, the negative lysosomal targeting (lysosomal avoidance) signals act at later stages of the endocytic pathway, most likely in late endosomal compartments.