Elucidating the Interaction between Pyridoxine 5′-Phosphate Oxidase and Dopa Decarboxylase: Activation of B6-Dependent Enzyme

Pyridoxal 5′-phosphate (PLP), the active form of vitamin B6, serves as a cofactor for scores of B6-dependent (PLP-dependent) enzymes involved in many cellular processes. One such B6 enzyme is dopa decarboxylase (DDC), which is required for the biosynthesis of key neurotransmitters, e.g., dopamine and serotonin. PLP-dependent enzymes are biosynthesized as apo-B6 enzymes and then converted to the catalytically active holo-B6 enzymes by Schiff base formation between the aldehyde of PLP and an active site lysine of the protein. In eukaryotes, PLP is made available to the B6 enzymes through the activity of the B6-salvage enzymes, pyridoxine 5′-phosphate oxidase (PNPO) and pyridoxal kinase (PLK). To minimize toxicity, the cell keeps the content of free PLP (unbound) very low through dephosphorylation and PLP feedback inhibition of PNPO and PLK. This has led to a proposed mechanism of complex formation between the B6-salvage enzymes and apo-B6 enzymes prior to the transfer of PLP, although such complexes are yet to be characterized at the atomic level, presumably due to their transient nature. A computational study, for the first time, was used to predict a likely PNPO and DDC complex, which suggested contact between the allosteric PLP tight-binding site on PNPO and the active site of DDC. Using isothermal calorimetry and/or surface plasmon resonance, we also show that PNPO binds both apoDDC and holoDDC with dissociation constants of 0.93 ± 0.07 μM and 2.59 ± 0.11 μM, respectively. Finally, in the presence of apoDDC, the tightly bound PLP on PNPO is transferred to apoDDC, resulting in the formation of about 35% holoDDC.     

A phase I/II trial of transurethral surgery combined with concurrent cisplatin, 5-fluorouracil and twice daily radiation followed by selective bladder preservation in operable patients with muscle invading bladder cancer

PURPOSE: We describe a protocol designed to evaluate the use of twice daily radiation used together with cisplatin and 5 fluorouracil (5-FU) in the treatment of operable transitional cell carcinoma of the bladder with potential bladder preservation. MATERIALS AND METHODS: A total of 18 consecutive patients with T2-T4a bladder tumors underwent as complete a transurethral resection as possible, which was visibly complete in 14 cases. They then received twice daily radiation and infusion cisplatin and 5-FU during an induction phase. No therapy was given for 3 weeks, following which patients were reevaluated cystoscopically. Cases of clinical complete response by biopsy and cytology were consolidated with further chemotherapy/radiation using the same chemotherapeutic agents and radiation schedule. Patients who had incomplete responses were advised to undergo an immediate radical cystectomy. Of the 18 patients 15 subsequently received 3 cycles of adjuvant chemotherapy, consisting of methotrexate, cisplatin and vinblastine. Median followup for the entire group is 32 months. RESULTS: Of the 18 patients 14 had no detectable tumor after induction therapy. Of the 4 patients with persistent tumor 2 underwent radical cystectomy and 2 refused cystectomy, 1 of whom was treated with partial cystectomy and the other with consolidation chemotherapy/radiation. The actuarial overall survival at 3 years was 83%. The chance of a patient being alive at 3 years with a native bladder was 78%. No patient required cystectomy for hematuria or bladder shrinkage. Three patients in whom superficial tumors developed were treated successfully with bacillus Calmette-Guerin. Small bowel obstruction in 1 case was corrected surgically. CONCLUSIONS: This pilot study demonstrates a high rate of response to this combined chemotherapy/radiation regimen in conjunction with a visibly complete transurethral resection. Reevaluation after a short induction phase allows for the early selection of patients with persistent disease for radical cystectomy.     

High-dose therapy with peripheral blood stem cell (PBSC) support using an innovative mobilization regimen in patients with high-risk primary or chemoresponsive metastatic breast cancers

BACKGROUND: Epidemiologic studies are necessary to determine the prevalence of allergic diseases. This varies widely depending on allergen preparations and patients studied. OBJECTIVE: To investigate the prevalence of atopic disease, skin test reactivity, total and specific IgE to common allergens, and other variables in a sample of students from Málaga, southern Spain. METHODS: Three hundred sixty-five students (age 17.9 +/- 1.18) were interviewed by an allergist. Skin prick tests were performed with Dermatophagoides pteronyssinus, Artemisia vulgaris, Plantago lanceolata, Chenopodium album, Olea europaea, Phleum pratense, Parietaria judaica, Cynodon dactylon, Alternaria tenuis, and cat dander. Total and specific IgE to D. pteronyssinus, Olea, and Parietaria were determined. RESULTS: Of all subjects studied, 19.9% suffered from rhinoconjunctivitis, 4.1% rhinoconjunctivitis plus asthma, 3.1% asthma alone, and 0.8% atopic dermatitis; 46.4% had a positive skin test to at least one allergen (28.2% to D. pteronyssinus, 20.4% to Olea, 13.8% to Phleum); and 43% had total IgE > 100 kU/L and 44.7% a family history of atopy. Allergic symptoms were strongly associated with skin test positivities and family allergic history. Patients with asthma or skin prick test positive had higher total IgE values than others (P < .01). There was a significant correlation between specific IgE values and wheal size in skin test. CONCLUSIONS: Our findings confirm the high prevalence of atopic diseases, and the close relationship of skin tests reactivity (or presence of specific IgE) to allergens with symptoms of asthma and rhinitis. The presence of a family history of allergic diseases influences the development of positive skin tests and atopic illness. Dermatophagoides pteronyssinus and pollen of Olea europaea were found to be the most common allergens.     

Genetic analysis of Drosophila larval optic nerve development

To identify genes necessary for establishing connections in the Drosophila sensory nervous system, we designed a screen for mutations affecting development of the larval visual system. The larval visual system has a simple and stereotypic morphology, can be recognized histologically by a variety of techniques, and is unnecessary for viability. Therefore, it provides an opportunity to identify genes involved in all stages of development of a simple, specific neuronal connection. By direct observation of the larval visual system in mutant embryos, we identified 24 mutations affecting its development; 13 of these are larval visual system-specific. These 13 mutations can be grouped phenotypically into five classes based on their effects on location, path or morphology of the larval visual system nerves and organs. These mutants and phenotypic classifications provide a context for further analysis of neuronal development, pathfinding and target recognition.     

Ca2+ and receptor-associated protein are independently required for proper folding and disulfide bond formation of the low density lipoprotein receptor-related protein

The low density lipoprotein receptor-related protein (LRP) is a cysteine-rich, multifunctional receptor that binds and endocytoses a diverse array of ligands. Recent studies have shown that a 39-kDa receptor-associated protein (RAP) facilitates the proper folding and subsequent trafficking of LRP within the early secretory pathway. In the current study, we have examined the potential role of Ca2+ and its relationship to RAP during LRP folding. We found that depletion of Ca2+ following either ionomycin or thapsigargin treatment significantly disrupts the folding process of LRP. The misfolded LRP molecules migrate as high molecular weight aggregates under nonreducing SDS-polyacrylamide gel electrophoresis, suggesting the formation of intermolecular disulfide bonds. This misfolding is reversible because misfolded LRP can be re-folded into functional receptor molecules upon Ca2+ restoration. Using an LRP minireceptor representing the fourth ligand binding domain of LRP, we also observed significant variation in the conformation of monomeric receptor upon Ca2+ depletion. The role of Ca2+ in LRP folding is independent from that of RAP because RAP remains bound to LRP and its minireceptor following Ca2+ depletion. Furthermore, Ca2+ depletion-induced LRP misfolding occurs in RAP-deficient cells. Taken together, these results clearly demonstrate that Ca2+ and RAP independently participate in LRP folding.     

Multiple conformations of physiological membrane-bound cytochrome c

One-tenth of cytochrome c (cyt c) remains bound to the inner mitochondrial membrane (IMM) at physiological ionic strength (I; i.e. , I approximately 150 mM), exhibiting decreased electron transport (ET) activity. We now show that this form of membrane-bound cyt c (MB-cyt c) can be obtained in vitro and that binding to membranes at low I generates an additional conformation with higher ET activity. This low I bound form of MB-cyt c (MBL-cyt c) exhibited intrinsic ET rates similar to those of electrostatically bound cyt c (EB-cyt c). The ET activity of IMM-bound MB-cyt c approached slowly that of MBL-cyt c or EB-cyt c, suggesting that MB-cyt c converts to MBL-cyt c while bound to IMM. When maintained at physiological I, both forms of MB-cyt c were released from the membrane, indicating that they convert to an EB-cyt c-like form. This process may be very dynamic in cellular mitochondria, as binding and release for both MB-cyt c forms increased considerably with temperature. I-Dependent binding of MB-cyt c does not require IMM, and it can be reproduced using large or small unilamellar vesicles (SUV). Using SUV-cyt c complexes, we characterized the secondary structure of MB-cyt c and MBL-cyt c by circular dichroism. Conformational analysis revealed that cyt c binding as MB-cyt c decreases its alpha-helical content (70-79%) and increases its beta-sheet up to 135%. The secondary structure of MBL-cyt c was similar to that of EB-cyt c and soluble cyt c, with a modest increase in beta-sheet. Taken together, our experiments suggest that physiological cyt c exists in soluble and membrane-bound conformations with similar ET activity, which may exchange very rapidly, and that soluble hydrophilic proteins can bind transiently to biomembranes.     

Analgesic efficacy of ibuprofen alone and in combination with codeine or caffeine in post-surgical pain: a meta-analysis

OBJECTIVE: To estimate the analgesic effect of ibuprofen and to test whether codeine and caffeine enhance its effect on post-surgical pain. METHOD: Systematic overview of the literature and meta-analysis of published randomised, controlled trials. RESULTS: Ibuprofen is effective in dental pain, episiotomy pain and other post-operative pain. There is a dose response relationship over the range 50-400 mg. The difference in total pain-relief score relative to placebo was 19-31%. On average, patients were over three times more likely to obtain moderate to excellent pain relief with ibuprofen than with placebo (response-rate ratio = 3.45) and the number needed to treat was 2.44. Codeine 60 mg enhanced the analgesic effect of ibuprofen 400 mg by about 8% in the total pain-relief scale, but it also increased its adverse effects. The additive effect of caffeine was inconsistent. CONCLUSION: Ibuprofen is an effective analgesic in post-operative pain. Codeine 60 mg adds to the analgesic effect of ibuprofen 400 mg. Any additive caffeine effect requires validation.     

Genomic amplification of a decoy receptor for Fas ligand in lung and colon cancer

Fas ligand (FasL) is produced by activated T cells and natural killer cells and it induces apoptosis (programmed cell death) in target cells through the death receptor Fas/Apol/CD95. One important role of FasL and Fas is to mediate immune-cytotoxic killing of cells that are potentially harmful to the organism, such as virus-infected or tumour cells. Here we report the discovery of a soluble decoy receptor, termed decoy receptor 3 (DcR3), that binds to FasL and inhibits FasL-induced apoptosis. The DcR3 gene was amplified in about half of 35 primary lung and colon tumours studied, and DcR3 messenger RNA was expressed in malignant tissue. Thus, certain tumours may escape FasL-dependent immune-cytotoxic attack by expressing a decoy receptor that blocks FasL.