Thyroid hormone regulation of cell migration and oxidative metabolism in polymorphonuclear leukocytes: clinical evidence in thyroidectomized subjects on thyroxine replacement therapy

Migration and superoxide anion (O2-) generation were studied in polymorphonuclear leukocytes (PMNs) from 14 athyreotic patients, previously treated by total thyroidectomy and radioiodine therapy for differentiated thyroid carcinoma, and from age- and sex-matched euthyroid healthy controls. Patients were studied twice: in hypothyroidism (visit 1) and after TSH-suppressive L-T4 replacement therapy (visit 2). Random migration and N-formyl-Met-Leu-Phe (fMLP) 0.1-microM induced chemotaxis were similar in cells from patients at both visit 1 and visit 2 and from healthy controls. On the contrary, resting O2- generation in cells from patients was significantly lower than control values, both at visit 1 and 2. At visit 1, fMLP 0.1 muM-induced O2- generation was significantly lower than control values, while phorbol-myristate acetate (PMA) 100-ng/ml induced O2- generation was similar in cells from patients and from controls. At visit 2 both responses increased, resulting in fMLP-induced O2- generation superimposable to control values and PMA-induced O2- generation significantly higher with respect to both visit 1 and cells from controls. In vitro exposure of PMNs from healthy subjects to L-T4 did not affect O2- generation in resting cells, and significantly increased that induced by fMLP or PMA only at high, supra-physiological concentrations. Neither TSH nor T3 had significant effects at any of the concentrations tested. The present results document the existence of a correlation between thyroid status and oxidative metabolism of human PMNs, which is however unlikely to depend upon a direct action of thyroid hormones on these cells.     

Synthesis of 8-Oxa analogues of norcocaine endowed with interesting cocaine-like activity.

In order to further explore the importance of cocaine's bridge nitrogen atom in binding to the dopamine transporter (DAT), we have synthesized the previously known racemic 8-oxa-norcocaines 3-6 in which the nitrogen atom has been replaced by oxygen. Additionally, to avoid incorrect interpretations of biological data that may stem from the use of racemic materials, several of these analogues were synthesized and tested in non-racemic form. (-)-8-Oxa-norcocaine (3) was found to bind to the cocaine recognition site and to inhibit the dopamine transporter with potencies only about 8-fold and 4-fold, respectively, less than those of norcocaine (2). (-)-8-Oxa-pseudonorcocaine (4) as well as (+)-8-oxa-norcocaine (3) were found to be comparable in activity to (-)-oxa-norcocaine. These pharmacological findings support our earlier suggestion that cocaine is likely to bind in its neutral form to the DAT.     

Time series in analysis of yerba-mate biennial growth modified by environment

To assess differences in the lag-effect pattern in the relationship between yerba-mate biennial growth and environmental factors, a time-series analysis was performed. A generalized Poisson regression model was used to control time trends, temperature, growing degree days (GDD), rainfalls and night length (NL). It was hypothesized that the active growth and growth pauses in yerba-mate are controlled endogenously and modified by environment, and that genders would respond differently to environmental modifications. The patterns in the lag effect from the distributed-lag models were similar to those of time-series models with meteorological data means with lag = 0. GDD and NL were principal factors affecting biennial yerba-mate shoot elongation and the number of green leaves of females grown in monoculture, besides their significant effects on metamer emission and leaf area in males grown in monoculture. NL also had a significant influence on shoot elongation and leaf area of both genders grown in forest understorey (FUS), indicating that yerba-mate growth is synchronized by an internal clock sensitive to temperature adjustments. The morphological plasticity and the adaptation efforts of yerba-mate were more pronounced in monoculture than in FUS. Sexual dimorphism was expressed—males were more sensitive to environmental changes than females, especially in monoculture. Growth modifications were much more intense when plants were grown in a cultivation system that is less like yerba-mate natural habitat (monoculture) than in one resembling its natural habitat (FUS). Our data support the ecological specialization theory.     

Molecular cloning and functional characterization of endogenous recombinant common marmoset monkey (Callithrix jacchus) follicle-stimulating hormone

Background Common marmoset monkeys (Callithrix jacchus) are readily used in biomedical research. However, superovulation for embryonic stem cell production and developmental research still remain difficult. Inexplicably, follicle‐stimulating hormone (FSH) as key player in superovulation has to be administered in extremely high dosages in this non‐human primate compared to human. Methods To evaluate whether marmoset FSH (cjFSH) is functionally more competent than its human homologue on the marmoset FSH receptor (cjFSHR), we established in vitro a homologous system characterizing homologous and recombinantly produced cjFSH. Results Upon stimulation of two cell lines stably expressing either the marmoset or the human FSH receptor (cj/hFSHR), respectively, the second messenger signaling of the activated receptor displayed no significant difference in ED₅₀ values. Thermostability of cjFSH was significantly prolonged by roughly 20% on both FSHRs. Conclusion High FSH dosage in marmoset superovulation cannot be explained by enhanced biopotency of the natural animal’s gonadotropin.     

Effects of cell-type specific leptin receptor mutation on leptin transport across the BBB

The functions of leptin receptors (LRs) are cell-type specific. At the blood-brain barrier, LRs mediate leptin transport that is essential for its CNS actions, and both endothelial and astrocytic LRs may be involved. To test this, we generated endothelia specific LR knockout (ELKO) and astrocyte specific LR knockout (ALKO) mice. ELKO mice were derived from a cross of Tie2-cre recombinase mice with LR-floxed mice, whereas ALKO mice were generated by a cross of GFAP-cre with LR-floxed mice, yielding mutant transmembrane LRs without signaling functions in endothelial cells and astrocytes, respectively. The ELKO mutation did not affect leptin half-life in blood or apparent influx rate to the brain and spinal cord, though there was an increase of brain parenchymal uptake of leptin after in situ brain perfusion. Similarly, the ALKO mutation did not affect blood-brain barrier permeation of leptin or its degradation in blood and brain. The results support our observation from cellular studies that membrane-bound truncated LRs are fully efficient in transporting leptin, and that basal levels of astrocytic LRs do not affect leptin transport across the endothelial monolayer. Nonetheless, the absence of leptin signaling at the BBB appears to enhance the availability of leptin to CNS parenchyma. The ELKO and ALKO mice provide new models to determine the dynamic regulation of leptin transport in metabolic and inflammatory disorders where cellular distribution of LRs is shifted.     

Identification of a Passiflora alata Curtis dimeric peptide showing identity with 2S albumins

Antifungal proteins and peptides, essential compounds for plant defense, have been isolated from several tissues of various plants. These proteins could be used as a natural alternative to control phytopathogenic fungi. In this report a heterodimeric antifungal protein named Pa-AFP1, showing higher identity with the 2S albumin family, was purified by using 70-100% ammonium sulfate saturation and further purification steps such as anionic exchange Q-Sepharose chromatography associated with HPLC reversed-phase C4 chromatography. Analysis by Tricine-SDS-PAGE revealed two peptidic molecular masses of approximately 4500 Da and 7000 Da, in the presence of β-mercaptoethanol, while by removing the reducing agent a single protein with molecular mass of about 11,500 Da was obtained. Moreover, dimer mass was confirmed by MALDI-TOF analyses (11,569.76 Da). The antifungal protein, named Pa-AFP1, efficiently inhibited the growth of filamentous fungi Colletotrichum gloeosporioides, and was added to a short list of 2S albumins with antimicrobial properties. Otherwise, this same peptide showed no activity toward bacteria and yeasts. In summary, this compound could be used in the future to develop biotechnological products for the control of phytopathogenic fungi.     

Bacterial chimeras and reversible phosphorylation: the work of Walther Stoeckenius

In 1971, Walther Stoeckenius discovered that Halobacterium halobium contains a purple pigment that is chemically similar to rhodopsin and works as a light-driven proton pump. This discovery set Stoeckenius on a research path centered on bacteriorhodopsin, which included the creation of a bovine-soybean-halobacteria chimera that was able to produce ATP when exposed to light and the discovery of a class of proteins that are phosphorylated in a light-dependent manner.Reconstitution of Purple Membrane Vesicles Catalyzing Light-driven Proton Uptake and Adenosine Triphosphate Formation (Racker, E., and Stoeckenius, W. (1974) J. Biol. Chem. 249, 662–663)Light-regulated Retinal-dependent Reversible Phosphorylation of Halobacterium Proteins (Spudich, J. L., and Stoeckenius, W. (1980) J. Biol. Chem. 255, 5501–5503)Walther Stoeckenius was born in 1921 in Giessen, Germany. He earned an M.D. degree from the University of Hamburg in 1950, after which he spent 18 months doing clinical work as an intern. In 1952, he began postdoctoral work at the Institute for Tropical Medicine in Hamburg, using electron microscopy to study the development of pox viruses. Two years later, he joined the Department of Pathology at the University of Hamburg as an assistant professor and became Docent for Pathology in 1958. At Hamburg, Stoeckenius continued to use electron microscopy to explore the fine structure of cells and the lipid membrane.In 1959, Stoeckenius left Germany to become a research associate in Keith Porter''s laboratory at Rockefeller University. After a few months, he became an assistant professor at Rockefeller, remaining there for 8 years and eventually becoming an associate professor. He continued to work on membrane structure, studying Halobacterium halobium, until he accepted a professorship at the University of California, San Francisco in 1967.In San Francisco, Stoeckenius focused more on biochemical techniques rather than electron microscopy. In collaboration with Dieter Oesterhelt, he discovered that H. halobium contains a purple pigment (bacteriorhodopsin) that is chemically similar to rhodopsin (1) and plays an important role in light energy storage in halobacteria, working as a light-driven proton pump (2).This discovery led to a collaboration with Journal of Biological Chemistry (JBC) Classic author Efraim Racker (3) in which Stoeckenius and Racker created a thoroughly unnatural vesicle. As reported in the first JBC Classic reprinted here, they used sonication to recombine membrane lipids from soybeans, bacteriorhodopsin from halobacteria, and ATPase from beef mitochondria. The resulting artificial vesicles were able to produce ATP when exposed to light. The chimeric vesicles also formed a simple model system for a biological proton pump capable of generating ATP from ADP and P_i.Stoeckenius continued to study bacteriorhodopsin and its light-driven proton uptake in bacteria. As reported in the second JBC Classic reprinted here, he discovered that phosphorylation is regulated by light absorbed by bacteriorhodopsin (4). Using [³²P]orthophosphate pulse labeling, Stoeckenius and John Spudich identified a class of phosphoproteins in H. halobium. Exposing labeled whole cells to light resulted in rapid dephosphorylation of two of the proteins, which were rapidly rephosphorylated upon darkening of the cells. The light sensitivity of the proteins was responsive to the presence of retinal, indicating that the dephosphorylation depended on rhodopsin-like (retinal-containing) photoreceptors.Stoeckenius currently is Professor Emeritus in the Department of Biochemistry and Biophysics and the Cardiovascular Research Institute at the University of California, San Francisco. He was elected to the National Academy of Sciences in 1978.     

Identification of a Napin-Like Peptide from Eugenia malaccensis L. Seeds with Inhibitory Activity Toward Staphylococcus aureus and Salmonella Enteritidis

This study aimed to purify and characterize peptides from the seeds of Eugenia malaccensis, L. (jambo) with inhibitory activity against the foodborne pathogens Staphylococcus aureus and Salmonella Enteritidis. Crude extract (CE), precipitate fraction 30–60 % and molecules between 3.5 and 10 kDa obtained from precipitate fraction 30–60 % (Em2) showed inhibitory activity against the tested bacterial strains. The highest antibacterial activity was observed for Em2 against S. aureus. The major peak eluted at approximately 30 % in an acetonitrile gradient in reverse-phase chromatography of Em2 (Em2-F1 to Em2-F19), and it showed the highest antibacterial activity, which was twofold higher against S. aureus than against S. Enteritidis. MALDI-ToF spectra of Em2-F18 revealed a molecular mass of 1,231.1 Da and the amino acid sequence showed high identity to the napin family. These findings report for the first time a napin-like peptide from E. malaccensis L. seeds with potential to be applied as a new anti-Staphylococcus molecule.