γ-Tocopherol biokinetics and transformation in humans

Background: The uptake and biotransformation of γ-tocopherol (γ-T) in humans is largely unknown. Using a stable isotope method we investigated these aspects of γ-T biology in healthy volunteers and their response to γ-T supplementation.

Methods: A single bolus of 100 mg of deuterium labeled γ-T acetate (d²-γ-TAC, 94% isotopic purity) was administered with a standard meal to 21 healthy subjects. Blood and urine (first morning void) were collected at baseline and a range of time points between 6 and 240 h post-supplemetation. The concentrations of d² and d⁰-γ-T in plasma and its major metabolite 2,7,8-trimethyl-2-(b-carboxyethyl)-6-hydroxychroman (-γ-CEHC) in plasma and urine were measured by GC-MS. In two subjects, the total urine volume was collected for 72 h post-supplementation. The effects of γ-T supplementation on α-T concentrations in plasma and α-T and γ-T metabolite formation were also assessed by HPLC or GC-MS analysis.

Results: At baseline, mean plasma α-T concentration was approximately 15 times higher than γ-T (28.3 vs. 1.9 µmol/l). In contrast, plasma γ-CEHC concentration (0.191 µmol/l) was 12 fold greater than α-CEHC (0.016 µmol/l) while in urine it was 3.5 fold lower (0.82 and 2.87 µmol, respectively) suggesting that the clearance of α-CEHC from plasma was more than 40 times that of γ-CEHC. After d²-γ-TAC administration, the d² forms of γ-T and γ-CEHC in plasma and urine increased, but with marked inter-individual variability, while the d⁰ species were hardly affected. Mean total concentrations of γ-T and γ-CEHC in plasma and urine peaked, respectively, between 0–9, 6–12 and 9–24 h post-supplementation with increases over baseline levels of 6–14 fold. All these parameters returned to baseline by 72 h. Following challenge, the total urinary excretion of d²-γ-T equivalents was approximately 7 mg. Baseline levels of γ-T correlated positively with the post-supplementation rise of (d⁰ + d²) – γ – T and γ-CEHC levels in plasma, but correlated negatively with urinary levels of (d⁰ + d²)-γ-CEHC. Supplementation with 100 mg γ-TAC had minimal influence on plasma concentrations of α-T and α-T-related metabolite formation and excretion.

Conclusions: Ingestion of 100mg of γ-TAC transiently increases plasma concentrations of γ-T as it undergoes sustained catabolism to CEHC without markedly influencing the pre-existing plasma pool of γ-T nor the concentration and metabolism of α-T. These pathways appear tightly regulated, most probably to keep high steady-state blood ratios α-T to γ-T and γ-CEHC to α-CEHC.     

Cdc42 and actin control polarized expression of TI-VAMP vesicles to neuronal growth cones and their fusion with the plasma membrane

Tetanus neurotoxin-insensitive vesicle-associated membrane protein (TI-VAMP)-mediated fusion of intracellular vesicles with the plasma membrane is crucial for neurite outgrowth, a pathway not requiring synaptobrevin-dependent exocytosis. Yet, it is not known how the TI-VAMP membrane trafficking pathway is regulated or how it is coordinated with cytoskeletal dynamics within the growth cone that guide neurite outgrowth. Here, we demonstrate that TI-VAMP, but not synaptobrevin 2, concentrates in the peripheral, F-actin-rich region of the growth cones of hippocampal neurons in primary culture. Its accumulation correlates with and depends upon the presence of F-actin. Moreover, acute stimulation of actin remodeling by homophilic activation of the adhesion molecule L1 induces a site-directed, actin-dependent recruitment of the TI-VAMP compartment. Expression of a dominant-positive mutant of Cdc42, a key regulator of cell polarity, stimulates formation of F-actin- and TI-VAMP-rich filopodia outside the growth cone. Furthermore, we report that Cdc42 activates exocytosis of pHLuorin tagged TI-VAMP in an actin-dependent manner. Collectively, our data suggest that Cdc42 and regulated assembly of the F-actin network control the accumulation and exocytosis of TI-VAMP-containing membrane vesicles in growth cones to coordinate membrane trafficking and actin remodeling during neurite outgrowth.     

Multiscale modeling of the cardiovascular system: application to the study of pulmonary and coronary perfusions in the univentricular circulation

The objective of this study is to compare the coronary and pulmonary blood flow dynamics resulting from two configurations of systemic-to-pulmonary artery shunts currently utilized during the Norwood procedure: the central (CS) and modified Blalock Taussig (MBTS) shunts. A lumped parameter model of the neonatal cardiovascular circulation and detailed 3-D models of the shunt based on the finite volume method were constructed. Shunt sizes of 3, 3.5 and 4 mm were considered. A multiscale approach was adopted to prescribe appropriate and realistic boundary conditions for the 3-D models of the Norwood circulation. Results showed that the average shunt flow rate is higher for the CS option than for the MBTS and that pulmonary flow increases with shunt size for both options. Cardiac output is higher for the CS option for all shunt sizes. Flow distribution between the left and the right pulmonary arteries is not completely balanced, although for the CS option the discrepancy is low (50-51% of the pulmonary flow to the right lung) while for the MBTS it is more pronounced with larger shunt sizes (51-54% to the left lung). The CS option favors perfusion to the right lung while the MBTS favors the left. In the CS option, a smaller percentage of aortic flow is distributed to the coronary circulation, while that percentage rises for the MBTS. These findings may have important implications for coronary blood flow and ventricular function.     

<Emphasis Type="Italic">Crambe tataria</Emphasis>: actions for ex situ conservation

In vitro micropropagation by direct organogenesis and somatic embryogenesis via callus was developed for Crambe tataria (Brassicaceae). C. tataria is an endemic species of the Pontic-Pannonic region, but it is also present in Italy, where it is localized in Friuli on a characteristic grassland formation, called “magredi”. C. tataria is regarded as an endangered species. Leaf and root explants were subjected to plant regulator treatments, which invoked different morphogenic responses. Leaf explants produced more callus than root explants and a higher amount of callus was obtained with 1 mg l⁻¹ 2,4-D in combination with 2 mg l⁻¹ Kin. Somatic embryogenesis was obtained in calli maintained in a delayed subculture regime on media containing BAP in combination with NAA. Root explants cultured with BAP combined with NAA developed adventitious rosette shoots. Shoots rooted on half-strength MS media, and the number of roots per plantlet and their length were heavily dependent on sucrose content. The in vitro regenerated plantlets were acclimatized ex vitro and a mean of 50% of the plantlets survived and showed a true-to-type growth habit. This study describes the development of two in vitro micropropagation protocols, via direct organogenesis and via embryogenesis from callus, that are the basis for the application of in vitro tools for the establishment of basal collections with representative genetic diversity and for the long-term storage of plant genetic material.     

Expression of adult fast pattern of acetylcholinesterase molecular forms by mouse satellite cells in culture

The pattern of acetylcholinesterase (AChE) molecular forms, obtained by sucrose gradient sedimentation, was studied at different in vitro developmental stages of myogenic cells isolated from adult mouse skeletal muscle. Only the globular forms were present in rapidly dividing satellite cells during the first days in culture. After myotube formation, a pattern similar to that described in mammalian fast-twitch skeletal muscle was observed. This pattern did not change during the following period in culture (up to 1 month) nor could it be modified by co-culturing with spinal cord motoneurons or by addition of brain-derived extracts. The internal-external localization of AChE molecular forms has been determined by the use of echothiophate iodide, a membrane-impermeant irreversible inhibitor of AChE. Echothiophate-treated cultures showed about 40% of both asymmetric and globular forms localized on the sarcolemma, with their active sites oriented outward. Analysis of culture medium from untreated cultures revealed the presence of both asymmetric and globular forms. When the same analysis was repeated on cultures of myoblasts derived from 16-day-old mouse embryos, the pattern of AChE forms was different. The myotubes derived from these cells exhibit a very small proportion of asymmetric form, which was not released into the medium. This pattern was not further modified during the following days of culture, nor by co-cultures with spinal cord motoneurons or by incubations with brain-derived extracts. Thus, the myotubes derived from myoblasts express in culture a clear phenotypic difference when compared to the corresponding myotubes from satellite cells, supporting the view that these two myogenic cells are endowed with different developmental programs.     

Skin in the game: Epidermal molt as a driver of long-distance migration in whales

Long-distance migration in whales has historically been described as an annual, round-trip movement between high-latitude, summer feeding grounds, and low-latitude, winter breeding areas, but there is no consensus about why whales travel to the tropics to breed. Between January 2009 and February 2016, we satellite-tagged 62 antarctic killer whales (Orcinus orca) of four different ecotypes, of which at least three made short-term (6–8 weeks), long-distance (maximum 11,000 km, round trip), essentially nonstop, migrations to warm waters (SST 20°C–24°C), and back. We previously suggested that antarctic killer whales could conserve body heat in subfreezing (to −1.9°C) waters by reducing blood flow to their skin, but that this might preclude normal (i.e., continuous) epidermal molt, and necessitate periodic trips to warm waters for routine skin maintenance (“skin molt migration,” SMM). In contrast to the century-old “feeding/breeding” migration paradigm, but consistent with a “feeding/molting” hypothesis, the current study provides additional evidence that deferred skin molt could be the main driver of long-distance migration for antarctic killer whales. Furthermore, we argue that for all whales that forage in polar latitudes and migrate to tropical waters, SMM might also allow them to exploit rich prey resources in a physiologically challenging environment and maintain healthy skin.     

Biophysical Characterization of Met-G-CSF: Effects of Different Site-Specific Mono-Pegylations on Protein Stability and Aggregation

The limited stability of proteins in vitro and in vivo reduces their conversion into effective biopharmaceuticals. To overcome this problem several strategies can be exploited, as the conjugation of the protein of interest with polyethylene glycol, in most cases, improves its stability and pharmacokinetics. In this work, we report a biophysical characterization of the non-pegylated and of two different site-specific mono-pegylated forms of recombinant human methionyl-granulocyte colony stimulating factor (Met-G-CSF), a protein used in chemotherapy and bone marrow transplantation. In particular, we found that the two mono-pegylations of Met-G-CSF at the N-terminal methionine and at glutamine 135 increase the protein thermal stability, reduce the aggregation propensity, preventing also protein precipitation, as revealed by circular dichroism (CD), Fourier transform infrared (FTIR), intrinsic fluorescence spectroscopies and dynamic light scattering (DLS). Interestingly, the two pegylation strategies were found to drastically reduce the polydispersity of Met-G-CSF, when incubated under conditions favouring protein aggregation, as indicated by DLS measurements. Our in vitro results are in agreement with preclinical studies, underlining that preliminary biophysical analyses, performed in the early stages of the development of new biopharmaceutical variants, might offer a useful tool for the identification of protein variants with improved therapeutic values.