Wiskott–Aldrich syndrome protein (WASP) is in a complex with WASP-interacting protein (WIP). WASP levels, but not mRNA levels, were severely diminished in T cells from WIP −/− mice and were increased by introduction of WIP in these cells. The WASP binding domain of WIP was shown to protect WASP from degradation by calpain in vitro . Treatment with the proteasome inhibitors MG132 and bortezomib increased WASP levels in T cells from WIP −/− mice and in T and B lymphocytes from two WAS patients with missense mutations (R86H and T45M) that disrupt WIP binding. The calpain inhibitor calpeptin increased WASP levels in activated T and B cells from the WASP patients, but not in primary T cells from the patients or from WIP −/− mice. Despite its ability to increase WASP levels proteasome inhibition did not correct the impaired IL-2 gene expression and low F-actin content in T cells from the R86H WAS patient. These results demonstrate that WIP stabilizes WASP and suggest that it may also be important for its function.

In order to determine the comparative sensitivity of two methods of detecting Blastocystis hominis and to investigate the seasonality of infection with this enteric protozoan parasite, the present study was conducted. In each of two 3-month periods representing winter, spring (February-April) and summer (July-September), 500 routine stool submissions were examined for B. hominis using microscopy following either formol-ether concentration or in vitro culture using Jones' medium. The organism was detected in 39 of the 1,000 samples investigated using the in vitro culture technique and in none of the samples using the formol-ether concentration technique. In 82% of the B. hominis-positive samples, no concurrent bacterial or parasitic pathogens were found, and diarrhoea was the most commonly recorded symptom among patients. Infection was more prevalent in summer than in winter/spring, occurring primarily in the 71-80-year age group. Cysts were detected in 20.5% of positive samples, but only following Ficoll-Paque concentration of formol-ether concentrates. Cyst excretion was more prevalent in summer than in winter/spring.

This paper elucidates the status of the different morphological forms of Blastocystis and reports the existence of thin- and thick-walled cysts in B. hominis on the basis of current experimental evidence. It is suggested that the thin-walled cysts are autoinfectious, leading to multiplication of the organism in the intestinal tract. The thick-walled cysts are responsible for external transmission via the faecal-oral route. A life cycle for B. hominis is postulated on the basis of these findings.

The amino acid prodrug of acyclovir (ACV), valacyclovir (VACV), is an effective antiherpetic drug. Systemic availability of ACV in humans is 3 to 5 times higher after oral administration of VACV. Enhanced bioavailability of VACV has been attributed to its carrier-mediated intestinal absorption via hPEPT1 peptide transporter followed by rapid and complete conversion to ACV. An earlier report suggested that the dipeptide ester prodrugs of ACV possess high affinity toward the intestinal oligopeptide transporter hPEPT1 and therefore seem to be promising candidates in the treatment of oral herpes virus infections. In the present study, we have examined the bioavailability of a series of dipeptide prodrugs of ACV after oral administration in Sprague-Dawley rats with cannulated jugular and portal veins. The area under plasma-concentration time curves expressed as minutes microgram milliliter Ϫ1 for total concentration of VACV (208.4 Ϯ 41.2), and the dipeptide prodrugs Gly-Val-ACV (GVACV) (416.1 Ϯ 140.9), Val-Val-ACV (VVACV) (147.7 Ϯ 89.3), and Val-Tyr-ACV (VYACV) (180.7 Ϯ 81.2) were significantly higher than that of ACV (21.2 Ϯ 5.2) upon intestinal absorption. Interestingly, the bioavailability of ACV after administration of GVACV was approximately 2-fold higher than VACV. There was significant metabolism by hepatic first pass effect of the dipeptide prodrugs as evident by the higher levels of ACV obtained after systemic absorption compared with intestinal absorption of GVACV and VVACV. The dipeptide prodrugs of ACV exhibited higher systemic availability of regenerated ACV upon oral administration and thus seem to be promising drug candidates in treatment of genital herpes infections.Peptide transporters PepT1 and PepT2 are perhaps the drug transporters that have captured the most recent attention in drug delivery. Small peptides such as di-and tripeptides are transported by PepT1 and PepT2 in intestinal and renal epithelial cells, respectively. Structure, function, mechanism, and substrate specificity of the peptide transporters have been extensively studied

Heparan sulfate 3-O-sulfotransferase 2 (HS3ST2), an enzyme mediating 3-O-sulfation of heparan sulfate (HS), is silenced by hypermethylation in breast cancer. As HS has an important co-receptor function for numerous signal transduction pathways, the phenotypical changes due to HS3ST2 reexpression were investigated in vitro using high and low invasive breast cancer cell lines. Compared to controls, highly invasive HS3ST2-expressing MDA-MB-231 cells showed enhanced Matrigel invasiveness, transendothelial migration and motility. Affymetrix screening and confirmatory real-time PCR and Western blotting analysis revealed increased expression of several matrix metalloproteinases, cadherin-11, E-cadherin and CEACAM-1, while protease inhibitor and annexin A10 expression were decreased. Low invasive HS3ST2 -expressing MCF-7 cells became even less invasive, with no change in gelatinolytic MMP activity. HS3ST2 expression increased HS-dependent basal and FGF2-specific signaling through the constitutively active p44/42 MAPK pathway in MDA-MB-231 cells. Increased MAPK activation was accompanied by upregulation of ß-catenin in MDA-MB-231, and of the transcription factor Tcf4 in both cell lines. Dysregulation of Tcf4-regulated ion transporters and increased cytosolic acidification were observed in HS3ST2-expressing MDA-MB-231 cells, which is a possible underlying cause of increased chemosensitivity towards doxorubicine and paclitaxel in these cells. This study provides the first in vitro evidence of the involvement of HS3ST2 in breast cancer cell invasion and chemosensitivity.Heparan sulfate (HS) proteoglycans (PGs) are proteins containing highly sulfated glycosaminoglycan (GAG) chains. HS is present in all cell types and tissues and functionally interacts with growth factors, tyrosine kinase receptors, matrix metalloproteinases (MMPs) and extracellular matrix (ECM) proteins to modulate cell adhesion, proliferation and motility.1-3 HSPGs do not only regulate physiological processes, such as organogenesis, angiogenesis, blood coagulation and

Analyses of the inhibition pattern of EACV transport suggests the involvement of a single transport system; namely, B(0,+). Design of amino acid prodrugs seems to be an attractive strategy to enhance the solubility of the otherwise poorly aqueous soluble compounds and also to afford a targeted and possibly enhanced delivery of the active drug.

Biogenesis of lysosomes depends in mammalian cells on the specific recognition and targeting of mannose 6-phosphate-containing lysosomal enzymes by two mannose 6-phosphate receptors (MPR46, MPR300), key components of the extensively studied receptor-mediated lysosomal sorting system in complex metazoans. In contrast, the biogenesis of lysosomes is poorly investigated in the less complex metazoan Drosophila melanogaster. We identified the novel type I transmembrane protein lysosomal enzyme receptor protein (LERP) with partial homology to the mammalian MPR300 encoded by Drosophila gene CG31072. LERP contains 5 lumenal repeats that share homology to the 15 lumenal repeats found in all identified MPR300. Four of the repeats display the P-lectin type pattern of conserved cysteine residues. However, the arginine residues identified to be essential for mannose 6-phosphate binding are not conserved. The recombinant LERP protein was expressed in mammalian cells and displayed an intracellular localization pattern similar to the mammalian MPR300. The LERP cytoplasmic domain shows highly conserved interactions with Drosophila and mammalian GGA adaptors known to mediate Golgi-endosome traffic of MPRs and other transmembrane cargo. Moreover, LERP rescues missorting of soluble lysosomal enzymes in MPR-deficient cells, giving strong evidence for a function that is equivalent to the mammalian counterpart. However, unlike the mammalian MPRs, LERP did not bind to the multimeric mannose 6-phosphate ligand phosphomannan. Thus ligand recognition by LERP does not depend on mannose 6-phosphate but may depend on a common feature present in mammalian lysosomal enzymes. Our data establish a potential important role for LERP in biogenesis of Drosophila lysosomes and suggest a GGA function also in the receptor-mediated lysosomal transport system in the fruit fly.

No abstract