Induction of porphobilinogen oxygenase and porphobilinogen deaminase in rat blood under conditions of erythropoietic stress

Porphobilinogen is the substrate of two enzymes: porphobilinogen deaminase and porphobilinogen-oxygenase. The first one transforms it into the metabolic precursors of heme and the second diverts it from this metabolic pathway by oxidizing porphobilinogen to 5-oxopyrrolinones. Rat blood is devoid of porphobilinogen-oxygenase under normal conditions while it carries porphobilinogen-deaminase activity. When the rats were submitted to hypoxia (p_O2 = 0.42 atm) for 18 days, the activity of porphobilinogen-oxygenase appeared at the tenth day of hypoxia and reached the maximum at the 14–16th day. It decreased to a half after 2 days (half-life of the enzyme) and disappeared after 4 days of return to normal oxygen pressure. Porphobilinogen-deaminase activity increased after the first day of hypoxia, reached a maximum at the 14–16th day and did not decrease to normal values until the 15th day after return to normal oxygen pressure. The activities of both prophobilinogen-oxygenase and porphobilinogen-deaminase were induced by administration of erythropoietin. When rats were made anaemic with phenylhydrazine, porphobilinogen-oxygenase activity also appeared in the blood cells. Although the reticulocyte concentration was higher when compared to that obtained under hypoxia, the activities of the oxygenase obtained under both conditions were comparable. Porphobilinogen-deaminase activity was always closely related to the reticulocyte content. The appearance of porphobilinogen-oxygenase under the described erythropoietic conditions was due to a de novo induction of the enzyme, as shown by its inhibition with actinomycin D and cycloheximide. Porphobilinogen-oxygenase as well as porphobilinogen-deaminase were present in the rat bone marrow under normal conditions. Their activities increased in phenylhydrazine treated rats. The properties and kinetics of porphobilinogen-oxygenase from the rat blood and bone marrow were determined and found to differ in several aspects.     

Recombinant expression of Ole e 6, a Cys-enriched pollen allergen, in Pichia pastoris yeast: detection of partial oxidation of methionine by NMR

Olive pollen is one of the main causes of allergy in Mediterranean countries. Ole e 6, an olive pollen allergen, is a small (5.8 kDa) and acidic protein (pI 4.2) and no homologous proteins have been isolated or characterized so far. Ole e 6 has been efficiently expressed in the methylotrophic yeast Pichia pastoris. The cDNA encoding Ole e 6 was inserted into the plasmid vector pPIC9 and overexpressed in GS115 yeast cells. The recombinant product was purified by size-exclusion chromatography followed by reverse-phase HPLC. N-terminal sequencing, amino acid composition analysis, CD, NMR, and IgG-binding experiments were employed to characterize the purified protein. NMR data revealed the oxidation of the methionine at position 28 in approximately 50% of the recombinant protein but, although this alters its electrophoretic behavior, it did not affect folding or IgG-binding properties of rOle e 6. The recombinant form of Ole e 6 expressed in P. pastoris can be employed for structural and biochemical studies.     

Correlation between hydrophobic properties and efficiency of carrier-mediated membrane transduction and apoptosis of a p53 C-terminal peptide

Two membrane transporters, the 17 amino acid (aa) oligopeptide penetratin derived from the homeodomain of Antennapedia (Ant) and an analogue of the basic domain of TAT (aa 47-57) (TAT-a) from HIV-1, were tested as carriers for a p53 C-terminal peptide (aa 361-382) into human breast cancer cells. The studies were performed to determine whether the membrane-transduction efficiency of membrane carriers: Ant, TAT or TAT analogue (TAT-a) correlated with peptide hydrophobic features. Peptide-sequence analysis clearly demonstrated that the Ant sequence and p53 peptide sequence (p53p) together created a peptide with enhanced hydrophobic characteristics; while the TAT or TAT analogue (TAT-a) and p53p sequence together created a peptide with significantly less hydrophobic qualities. The degree of hydrophobic moment and helical wheel plots for these peptides correlated directly with their ability to transduce the p53 peptide. Western blot analysis revealed that Ant was able to transduce p53 C-terminal peptide into human breast cancer cells as a highly efficient membrane transporter. Compared to Ant, TAT-a fused to the C-terminus of p53 peptide (p53p-TAT-a) was a less efficient carrier into these cells under the conditions of our study. Additionally, N-terminal linked TAT-a to p53p (TAT-a-p53p) showed even lower efficiency as a transporter than p53-TAT-a. Apoptosis assays showed that the p53 peptide, fused at its C-terminus to Ant (p53p-Ant), induced a higher percentage of apoptotic cells in human breast cancer cell lines expressing mutant or wild-type p53 as compared to p53 peptide fused at its C-terminus to the TAT-a sequence (p53p-TAT-a) or when fused at the N-terminus to TAT-a (TAT-a-p53p). These data suggested a direct correlation between hydrophobic characteristics and efficiency as a transporter. Sequence study, using hydrophobic moment and helical wheel analyses, may be useful predictive tools for choosing the best carrier for a peptide.     

Neo-clerodane diterpenoids from Croton schiedeanus

Two new neo-clerodane type furano diterpenoids were isolated from the aerial part of Croton schiedeanus, besides the clerodane diterpenes cis- and trans-dehydrocrotonin, previously isolated from other species of Croton. Structural elucidation was achieved on basis of extensive NMR experiments, including X-ray diffraction analysis and molecular mechanics calculations. The previously known flavonoids ayanin and quercetin-3,7-dimethyl ether were also obtained from the extract of this plant.     

A Social-Ecological View of Barriers and Facilitators for HIV Treatment Adherence: Interviews with Puerto Rican HIV Patients

Purpose

To identify perceived barriers and facilitators for HAART adherence among people living with HIV/AIDS in Southern Puerto Rico using a Social Ecological framework.

Patients and Methods

Individual in-depths interviews were conducted with 12 HIV patients with a history of HAART non-adherence. Interviews were audio-taped and transcribed. Content analysis was performed for each transcribed interview by three independent coders using a codebook. Using Atlas TI, super-codes and families were generated to facilitate the categorization tree as well as grounded analyses and density estimates

Results

Most participants reported a monthly income of $500 or less (n = 7), a high school education level (n = 7), being unemployed (n = 9) and being recipients of government health insurance (n = 11). Three out of six women reported living alone with their children and most men informed living with their parents or other relatives (n = 4). For the grounded analyses, the top four sub-categories linked to high number of quotations were mental health barriers (G = 32) followed by treatment regimen (G = 28), health system (G = 24) and interpersonal relations (G = 16). The top four sub-categories linked to high number of codes are treatment regimen (D = 4), health status perception (D = 3), interpersonal relations (D = 3) and health system (D = 3).

Conclusion

The results of this study suggest the interconnection of HIV treatment adherence barriers at various system levels. Future studies on HIV treatment barriers should explore these interactions and investigate the possible synergistic effect on non-adherent behavior     

Synthesis of PEGylated lactose analogs for inhibition studies on <Emphasis Type="Italic">T.cruzi</Emphasis> trans-sialidase

Trypanosoma cruzi, the agent of Chagas disease, expresses a unique enzyme, the trans-sialidase (TcTS) involved in the transfer of sialic acid from host glycoconjugates to mucins of the parasite. The enzyme is shed to the medium and may affect the immune system of the host. We have previously described that lactose derivatives effectively inhibited the transfer of sialic acid to N-acetyllactosamine. Lactitol also prevented the apoptosis caused by the TcTS, although it is rapidly eliminated from the circulatory system. In this paper we report covalent conjugation of polyethylene glycol (PEG) with lactose, lactobionolactone and benzyl β-D-galactopyranosyl-(1→6)-2-amino-2-deoxy-α-D-glucopyranoside (1) with the hope to improve the bioavailability, though retaining their inhibitory properties. Different conjugation methods have been used and the behavior of the PEGylated products in the TcTS reaction was studied.