Soft X-Ray Second Harmonic Generation as an Interfacial Probe.

Nonlinear optical processes at soft x-ray wavelengths have remained largely unexplored due to the lack of available light sources with the requisite intensity and coherence. Here we report the observation of soft x-ray second harmonic generation near the carbon K edge (∼284 eV) in graphite thin films generated by high intensity, coherent soft x-ray pulses at the FERMI free electron laser. Our experimental results and accompanying first-principles theoretical analysis highlight the effect of resonant enhancement above the carbon K edge and show the technique to be interfacially sensitive in a centrosymmetric sample with second harmonic intensity arising primarily from the first atomic layer at the open surface. This technique and the associated theoretical framework demonstrate the ability to selectively probe interfaces, including those that are buried, with elemental specificity, providing a new tool for a range of scientific problems.

In human granulosa cells from small antral follicles, androgen receptor mRNA and androgen levels in follicular fluid correlate with FSH receptor mRNA.

Human small antral follicles (diameter 3-9 mm) were obtained from ovaries surgically removed for fertility preservation. From the individual aspirated follicles, granulosa cells and the corresponding follicular fluid were isolated in 64 follicles, of which 55 were available for mRNA analysis (24 women). Expressions of androgen receptor (AR) mRNA levels in granulosa cells, and of androstenedione and testosterone in follicular fluid, were correlated to the expression of the FSH receptor (FSHR), LH receptor (LHR), CYP19 and anti-Müllerian Hormone-receptor II (AMHRII) mRNA in the granulosa cells and to the follicular fluid concentrations of AMH, inhibin-B, progesterone and estradiol. AR mRNA expression in granulosa cells and the follicular fluid content of androgens both showed a highly significant positive association with the expression of FSHR mRNA in granulosa cells. AR mRNA expression also correlated significantly with the expression of AMHRII, but did not correlate with any of the hormones in the follicular fluid. These data demonstrate an intimate association between AR expression in immature granulosa cells, and the expression of FSHR in normal small human antral follicles and between the follicular fluid levels of androgen and FSHR expression. This suggests that follicular sensitivity towards FSH stimulation may be augmented by stimulation of androgens via the AR.

Synergistic effects of a cremophor EL drug delivery system and its U0126 cargo in an ex vivo model.

Neuroprotection has proven clinically unsuccessful in subarachnoid hemorrhage. We believe that this is because the major component in the early damage pathway, the vascular wall, has not been given the necessary focus. U0126 is a potent inhibitor of vascular phenotypical changes, exemplified by functional endothelin B (ETB) receptor upregulation. The current study aimed to determine the optimal dose of U0126 ex vivo and test the toxicology of this dose in vivo. To find the optimal dose and test a suitable in vivo delivery system, we applied an ex vivo model of blood flow cessation and investigated functional ETB receptor upregulation (using a specific agonist) as the primary endpoint. The secondary endpoint was depolarization-induced contractility assessed by 60 mM K+ stimuli. Furthermore, an in vivo toxicology study was performed on the optimal selected doses. U0126 (10 µM) had a strong effect on the prevention of functional ETB receptor contractility, combined with minimal effect on the depolarization-induced contractility. When cremophor EL was chosen for drug delivery, it had an inhibitory and additive effect (combined with U0126) on the ETB receptor contractility. Hence, 10 µM U0126 in 0.5% cremophor EL seems to be a dose that will be close to the maximal inhibition observed ex vivo on basilar arteries, without exhibiting side effects in the toxicology studies. U0126 and cremophor EL are well tolerated at doses that have effect on ETB receptor upregulation. Cremophor EL has an additional positive effect, preventing functional ETB receptor upregulation, making it suitable as a drug delivery system.

The E3 ubiquitin ligase SMURF1 regulates cell-fate specification and outflow tract septation during mammalian heart development.

Smad ubiquitin regulatory factor 1 (SMURF1) is a HECT-type E3 ubiquitin ligase that plays a critical role in vertebrate development by regulating planar cell polarity (PCP) signaling and convergent extension (CE). Here we show that SMURF1 is involved in mammalian heart development. We find that SMURF1 is highly expressed in outflow tract cushion mesenchyme and Smurf1-/- mouse embryos show delayed outflow tract septation. SMURF1 is expressed in smooth muscle cells of the coronary arteries and great vessels. Thickness of the aortic smooth muscle cell layer is reduced in Smurf1-/- mouse embryos. We show that SMURF1 is a negative regulator of cardiomyogenesis and a positive regulator of smooth muscle cell and cardiac fibroblast differentiation, indicating that SMURF1 is important for cell-type specification during heart development. Finally, we provide evidence that SMURF1 localizes at the primary cilium where it may regulate bone morphogenetic protein (BMP) signaling, which controls the initial phase of cardiomyocyte differentiation. In summary, our results demonstrate that SMURF1 is a critical regulator of outflow tract septation and cell-type specification during heart development, and that these effects may in part be mediated via control of cilium-associated BMP signaling.

Mechanisms controlling death, survival and proliferation in a model unicellular eukaryote Tetrahymena thermophila.

Below a critical cell density of 750 cells ml(-1), and in a manner familiar throughout much of cell culture technology, Tetrahymena thermophila die within a few hours despite being supported by a nutritionally complete synthetic medium, SSM, in which a supracritical inoculum (1000 cells ml(-1)) nevertheless proliferates and quickly reaches 1 x 10(6) cells ml(-1). The kinetics of cell death, and the conditions required to keep cells alive at and below the critical density have now been more fully investigated. Interestingly, cell death follows first order kinetics, with a half-life of less than two hours at 250 cells ml(-1). Survival can be extended by an order of magnitude, however, when protein synthesis is reduced by inoculation of cells at this density in: (a)Tris/HCI-buffer;(b) SSM deficient in an essential amino acid (arginine or phenylalanine); or, (c) SSM containing cycloheximide. In the presence of actinomycin D, the critical density required for proliferation can be lowered to 100 cells ml(-1). These results are discussed in relation to the capacity of Tetrahymenaio produce and release signal molecules (loosely referred to as growth factors), which need to be present above a certain threshold level before proliferation occurs. The evidence for the demise of cells at low density being active - in terms of requirement for, or dependency on, new transcriptional and translational processes - is discussed, along with more general implications of the findings for the control of cell death in populations of 'free-living' unicellular organisms in culture compared with their normal habitats.

Signaling in unicellular eukaryotes.

Aspects of intercellular and intracellular signaling systems in cell survival, proliferation, differentiation, chemosensory behavior, and programmed cell death in free-living unicellular eukaryotes have been reviewed. Comparisons have been made with both bacteria and metazoa. The central organisms were flagellates (Trypanosoma, Leishmania, and Crithidia), slime molds (Dictyostelium), yeast cells (Saccharomyces cerevisiae), and ciliates (Paramecium, Euplotes, and Tetrahymena). There are two novel aspects in this review. First, cellular responses are viewed in an evolutionary perspective, rather than from the more prevailing one, in which the unicellular eukaryotes are seen by the mammalian organisms. Second, results obtained with cell cultures in minimal, chemically defined nutrient media at low cell densities where intercellular signaling is strongly reduced are discussed. These results shed light on control mechanisms and their cooperation inside the living cell. Intracellular systems have many common features in unicellular and multicellular organisms.

Physiological studies on the effect of Ca2+ on the duration of the lag phase of Saccharomyces cerevisiae.

Cell multiplication and growth of Saccharomyces cerevisiae were followed in 2-ml test tubes containing Wickerham's synthetic medium or very dilute synthetic media supplemented in various ways. The ability of the cell cultures to leave the lag phase and enter the exponential phase of growth was investigated. Multiplication was assessed by microscopical observation. The results showed great differences in times required for the cultures to leave the lag phases and begin multiplication. In Wickerham's medium, all cultures grew well 6 h after inoculation. In the dilute medium, several days elapsed before all the cultures grew. These cultures went into exponential growth with approximately first order kinetics. In the unsupplemented medium, the 'half-lives' in the lag phase were about 28 h. Addition of either Ca2+ or Ca2+ plus A23187 (calcimycin) reduced the half-lives to 10 and 6 h, respectively. The doubling times in the exponential phases of growth were not shortened by these additions. We suggest that Ca2+ plays a crucial role as a signal to switch on the mode of cell proliferation in S. cerevisiae.

Cell survival and multiplication. The overriding need for signals: from unicellular to multicellular systems.

There are clear similarities in the control mechanisms for cell survival and multiplication in the two eukaryotes, the ciliate Tetrahymena thermophila and the yeast, Saccharomyces cerevisiae. Cell multiplication in both organisms is activated by the same compounds (phorbol esters, diacylglycerol, tetrapyrroles, etc.). These compounds also affect cell multiplication and other activities in mammalian cell systems. This homology in control mechanisms in two distinct groups of unicellular eukaryotes on the one hand, and in cells from multicellular animals on the other, leads us to propose that these cytoplasmic control mechanisms for cell survival and multiplication originated in the unicellular eukaryotes.

Origins of signalling and memory: matters of life versus death.

This review focuses on the principles in cell-cell communication and cellular ability to respond to external chemical changes which have been so crucial for the development of life on planet Earth. We now know that the capacity of free-living organisms which evolved more than a billion years ago to respond to intercellular signal molecules, originating either from themselves or from other sources in their vicinity, is so similar possibly even more sophisticated - to that of the cells in our own body, and these findings have had a major impact on our struggle to understand how life has evolved and how it can be maintained. Attention is drawn to the very important topic of mechanisms in cell death, being seen as an aggressive and very powerful instrument in the continuance of life and ability of life to proliferate into a plethora of new species, and use insulin-related material as our paradigm. Such signal molecules (hormones) may have played a major role in cellular maintenance throughout evolution.