Hypotonically induced whole-cell currents in A6 cells: relationship with cell volume and cytoplasmic Ca2+

We investigated changes in whole-cell currents, cell volume, and intracellular calcium concentration ([Ca2+]i) during hypotonic stimulation in whole-cell clamped cultured amphibian renal cells (A6 cells). Upon being exposed to hypotonic solution (80% osmolality), the A6 cells swelled and peaked in the first 5 min, which was followed by a progressive decrease in cell volume termed regulatory volume decrease (RVD). Following the cell swelling, there were large increases in both outward- and inward-currents, which seemed to be carried by K+ efflux and Cl- efflux, respectively. A K+ channel blocker (TEA or quinine) or a Cl- channel blocker (NPPB or SITS) significantly inhibited both currents and RVD, suggesting that the inward- and outward-currents are highly correlated with each other and essential to RVD. Hypotonic stimulation also induced a transient [Ca2+]i increase, of which the time course was essentially similar to that of the currents. When internal and external Ca2+ were deprived to eliminate the Ca2+ transient increase, whole-cell currents and RVD were strongly inhibited. On the other hand, channel blockers TEA and NPPB, which inhibited whole-cell currents and RVD, did not inhibit the [Ca2+]i increase. It is concluded that hypotonic stimulation to A6 cells first induces cell swelling, which is followed by [Ca2+]i increase that leads to the coactivation of K+ and Cl- channels. This coactivation may accelerate K+ and Cl- effluxes, resulting in RVD.     

Production of active recombinant eIF5A: reconstitution in E.coli of eukaryotic hypusine modification of eIF5A by its coexpression with modifying enzymes

Eukaryotic translation initiation factor 5A (eIF5A) is the only cellular protein that contains the polyamine-modified lysine, hypusine [N(ε)-(4-amino-2-hydroxybutyl)lysine]. Hypusine occurs only in eukaryotes and certain archaea, but not in eubacteria. It is formed post-translationally by two consecutive enzymatic reactions catalyzed by deoxyhypusine synthase (DHS) and deoxyhypusine hydroxylase (DOHH). Hypusine modification is essential for the activity of eIF5A and for eukaryotic cell proliferation. eIF5A binds to the ribosome and stimulates translation in a hypusine-dependent manner, but its mode of action in translation is not well understood. Since quantities of highly pure hypusine-modified eIF5A is desired for structural studies as well as for determination of its binding sites on the ribosome, we have used a polycistronic vector, pST39, to express eIF5A alone, or to co-express human eIF5A-1 with DHS or with both DHS and DOHH in Escherichia coli cells, to engineer recombinant proteins, unmodified eIF5A, deoxyhypusine- or hypusine-modified eIF5A. We have accomplished production of three different forms of recombinant eIF5A in high quantity and purity. The recombinant hypusine-modified eIF5A was as active in methionyl-puromycin synthesis as the native, eIF5A (hypusine form) purified from mammalian tissue. The recombinant eIF5A proteins will be useful tools in future structure/function and the mechanism studies in translation.     

Tunable te-CO2 laser-pumped formaldehyde far-infrared lasers: Offsets,assignment, and superfluorescence

Sixty and three absorption transitions in D₂CO and H₂CO, respectively, have produced a number of far infrared laser lines when they are pumped by an etalon-tuned TE-CO₂ laser. Almost all the absorption transitions pumped previously by a free runing TE-CO₂ laser have been efficiently pumped by the etalon tuned CO₂ laser and found to have offset within ±500 MHz from the line-center of the relevant CO₂ pump lines. 22 (1) absorption and 63 (4) emission lines of D₂CO (H₂CO) are assigned. Some of these lines have generated superfluorescence. In paticular, the D₂CO 319-μm line pumped by CO₂-9P(32) delivered an output energy of approximately one half that of the well-known D₂O 66-μm. It is shown that a large electric dipole moment and an appreciable amount of fractional population in the lower level of the pump transition of this line are responsible to the superfluorescence.     

Actin flow and talin dynamics govern rigidity sensing in actin–integrin linkage through talin extension

At cell–substrate adhesion sites, the linkage between actin filaments and integrin is regulated by mechanical stiffness of the substrate. Of potential molecular regulators, the linker proteins talin and vinculin are of particular interest because mechanical extension of talin induces vinculin binding with talin, which reinforces the actin–integrin linkage. For understanding the molecular and biophysical mechanism of rigidity sensing at cell–substrate adhesion sites, we constructed a simple physical model to examine a role of talin extension in the stiffness-dependent regulation of actin–integrin linkage. We show that talin molecules linking between retrograding actin filaments and substrate-bound integrin are extended in a manner dependent on substrate stiffness. The model predicts that, in adhesion complexes containing ≈30 talin links, talin is extended enough for vinculin binding when the substrate is stiffer than 1 kPa. The lifetime of talin links needs to be 2–5 s to achieve an appropriate response of talin extension against substrate stiffness. Furthermore, changes in actin velocity drastically shift the range of substrate stiffness that induces talin–vinculin binding. Our results suggest that talin extension is a key step in sensing and responding to substrate stiffness at cell adhesion sites.     

Up-regulation of integrin beta 3 expression by cyclic stretch in human umbilical endothelial cells

OBJECTIVE: Chronic fatigue syndrome (CFS) has been hypothesized to result from immune activation. We examined the role of serum markers of inflammation and immune activation among patients with CFS and in those with chronic fatigue (CF) not meeting the case definition. METHODS: Assays for soluble interleukin 2 (IL-2) receptor, IL-6, C-reactive protein, beta 2-microglobulin, and neopterin were performed in 153 fatigued patients in a referral clinic. Patients were classified according to whether they met criteria for CFS, reported onset of illness with a viral syndrome or had a temperature > 37.5 degrees C on examination. RESULTS: Compared to control subjects, mean concentrations of C-reactive protein, beta 2-microglobulin, and neopterin were higher in patients with CFS (p < or = 0.01) and CF (p < or = 0.01). Results did not distinguish CFS from CF. IL-6 was elevated among febrile patients compared to those without this finding (p < or = 0.001), but other consistent differences between patient subgroups were not observed. The presence of several markers was highly correlated (p < 0.01). CONCLUSION: Our findings that levels of several markers were significantly correlated points to a subset of patients with immune system activation. Whether this phenomenon reflects an intercurrent, transient, common condition, such as an upper respiratory infection, or is the result of an ongoing illness associated process is unknown. Overall, serum markers of inflammation and immune activation are of limited diagnostic usefulness in the evaluation of patients with CSF and CF.     

Nonlinear level crossing in the infrared transitions of optically pumped far infrared laser media

In conjunction with the Stark field induced power enhancement in optically pumped far infrared lasers, absorption transitions in a degenerate two level system under the influence of an weak static Stark field have been analyed. A third order theory to the density matrix formalism leads to zero field level crossing signals for absorption in rate equation approximation and in Doppler limit. For fast relaxation among Zeeman sublevels, the zero field level crossing signal arises from population effect. Optoacoustic measurements have been carried out on the ν₅,^qQ(16,8) absorption line of CH₃OH which produces the familiar 119-μm laser with the CO₂ 9P(36) pump line. The optoacoustic absorption signals observed are well reproduced by the theoretical expression.     

Similarity in growth of output power of cw far infrared lasers

By means of intracavity Stark-pulse technique, growth of output power of an optically pumped cw CH₃OH 119-μm laser has been observed for various pressures of the active medium and for various input optical powers. Comparison of the results obtained in this work with those of a previous report on a HCOOH laser suggests that there is similarity relation in growth of output power. The similarity relation in a two-level laser is discussed.     

Intramembrane structure of the sensory axon terminals in bullfrog muscle spindles

Much physiologic and morphologic research has been done into the sensory mechanism of the frog muscle spindle. However, no freeze-fracture study has described in detail the shape and intramembrane structure of the nonmyelinated sensory axon terminals of the frog muscle spindle. In this study, muscle spindles were isolated from the red part of bullfrog semitendinous muscles. Chemically fixed spindles were subjected to freeze fracturing. The sensory axon endings were reconstructed, and the size and density of intramembrane particles (IMPs) were measured along the sensory nerve endings. The axon terminals had four distinctive parts: parent trunks (>0.5 microm in diameter), primary branches (0.15-0.5 microm), terminal branches (<0.1 pm), and varicosities (0.02-0.5 microm). IMPs ranged from 5 nm to 21 nm in diameter and were present in the intramembrane space of the plasma membrane all throughout the nonmyelinated sensory nerve endings. Mean IMP sizes in the protoplasmic face (PF) and the external face (EF), respectively, were 8.1 nm and 8.4 nm in the parent trunks, 8.8 nm and 8.8 nm in the primary branches, 9.4 nm and 9.0 nm in the varicosities, and 8.7 nm and 8.7 nm in the terminal branches. Mean IMP size in the PF was smallest in the parent trunk and largest in the varicosity. Mean IMP densities (numbers of IMPs per microm2) in the PF and the EF, respectively, were 2,500 and 700 in the parent trunks, 2,200 and 500 in the primary branches, 1,700 and 400 in the varicosities, and 1,000 and 300 in the terminal branches. Density decreased with the tapering of the axon terminal, with IMPs distributed evenly in the PF and the EF. The characteristic intramembrane structure of sensory nerve endings is discussed.     

Electric field effects on an optically-pumped HCOOH FIR laser and Zeeman laser theory

Electric field effects have been investigated on the output power of six far-infrared (FIR) laser lines from H¹²COOH optically-pumped by a CO₂ laser with its polarization arranged perpendicular to the Stark field. Optoacoustic signals observed on the pump lines were hardly affected by the applied electric field up to 0.6 kV/cm. By neglecting the electric field effects on the pump transitions, Zeeman laser theory has been applied to the FIR laser transitions. Numerical calculation predicts the observed FIR output power as a function of electric field. Experessions for oscillation frequency and intensity in homogeneous limit are given, which may be applicable to any FIR Stark laser so far as the pump transition is free from electric field effects.