Suppressor cell-inducing factor: a new lymphokine secreted by a natural suppressor cell line with natural cytotoxic activity. I. Purification to apparent homogeneity and initial characterization of suppressor cell-inducing factor

The lymphokine suppressor cell-inducing factor (SIF), obtained from 15 liters of serum-free culture supernatants of the natural suppressor cell line, M1-A5, has been purified to apparent homogeneity by a combination of gel filtration, ion exchange chromatography, and reverse-phase-HPLC. Purity of SIF was assessed by the migration of the factor as a single band on SDS-PAGE, and the elution from reverse-phase-HPLC column as a single and sharp peak. SIF activity was retained after both procedures. Two protein factors with SIF activity were isolated from M1-A5 culture supernatants. The first protein factor (SIF alpha) had a Mr of 43 kDa, and the second protein factor (SIF beta) had a Mr of 6 kDa. Final purification of SIF alpha yielded 5 micrograms protein with specific activity of 4 x 10(6) U/mg protein. Final purification of SIF beta yielded 40 micrograms protein with specific activity of 7.5 x 10(7) U/mg protein. The relationship between SIF alpha and SIF beta, as well as the relationship with other suppressor factors, will be addressed.     

The use of a chloride-sensitive fluorescent probe to measure chloride transport in isolated tonoplast vesicles

A fluorescence method for the direct measurement of Cl^- transport in isolated tonoplast vesicles is described. This technique utilises the Cl^--sensitive fluorescent compound, 6-methoxy-1-(3-sulfonatopropyl)quinolinium (SPQ). This is a water-soluble compound with excitation and emission wavelengths of 350 and 440 nm, respectively. Its fluorescence is quenched by Cl^-, Br^-, I^-, SCN^-, NO ₂ ^- and tetraphenylborate but not by NO ₃ ^- , SO ₄ ^2- , iminodiacetate or malate. These effects are independent of pH. This compound was loaded into tonoplast vesicles from red beet (Beta vulgaris L.) storage roots or from barley (Hordeum vulgare L.) roots by incubation at 37° C and the external probe was then removed by repeated centrifugation of the vesicles in SPQ-free medium. In this way a large proportion of the observed fluorescence signal was from the interior of the vesicles, and its quenching could be used to monitor, quantitatively, and in real time, the intravesicular Cl^- concentration. In this paper we describe some of the problems encountered in using this probe to measure Cl^- transport in tonoplast vesicles, how these were overcome and some characteristics of Cl^- transport at the tonoplast as measured by the probe.Abbreviations and symbols BTP 1,3-bis[tris(hydroxymethyl)-methylamino-propane - DTT dithiothreitol - membrane potential - pH pH gradient - PPase inorganic pyrophosphatase - PPi inorganic pyrophosphate - SPQ 6-methoxy-1-(3-sulfonatopropyl)quinolinium - Tricine N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine     

A method for the intravital measurement of interspinous kinematics.

A novel non-radiographic technique for objectively quantifying quasi-static or dynamic intervertebral motion of a spinal motion segment in vivo in human subjects is presented here. The intervertebral motion device (IMD) is an instrumented linkage transducer system which can continuously measure over time two-dimensional sagittal plane rigid-body motion. Three custom-built omega-shaped displacement transducers are utilized. The IMD is rigidly fixed to the spinous processes of the lumbar motion segment by means of two intraosseous pins. Knowing the mechanoelectrical behavior and geometric configuration of the IMD, the relative spatial motion between the vertebral bodies can be resolved into sagittal rotation, axial translation, and anterior-posterior shear translation. Static calibrations of the IMD in the ranges of +/- 4 degrees rotation and +/- 4 mm translation determined the absolute maximum errors to be 0.2 degree and 0.07 mm for rotation and translation measurements, respectively, with corresponding variances of 0.1 degrees and 0.03 mm. For use in the vibration environment, negligible motion artifact content was detected in the IMD output signals when excited at discrete frequencies of 5.0 and 8.0 Hz. The first natural frequency of the IMD, specific for this design, was measured at 16.25 Hz. This technique may be used to study in vivo the spinal kinematics in healthy lumbar motion segments and in patients suspected of having segmental instability, and can perhaps be of clinical diagnostic significance.     

Motional narrowing of the 2H NMR spectra near the chain melting transition of phospholipid/D2O mixtures

The reduction in spectral splitting, or motional narrowing, of the deuterium spectra of D₂O/phos-pholipid mixtures near the main chain melting phase transition was studied for palmitoyloleoylphosphatidylcholine (POPC), palmitoyloleoylphosphatidylethanolamine (POPE) and equimolar mixtures of the two at 10% hydration. For POPC the splitting was about 1700 Hz in both the fluid and gel phases, dropping to zero near the phase transition (as reported previously). For POPE the splitting remained approximately constant above the phase transition. Below the phase transition the spectrum showed a single broad line whose linewidth varied between 100 Hz and 800 Hz. This was interpreted as being due to small domains of water within a weakly hydrated crystal. POPC:POPE (1:1) samples exhibited motional narrowing behaviour similar to that for POPC except that the splitting above the phase transition was approximately twice that below the transition. The relatively broad temperature range (20 K) of the transition is explained using a simple physical model involving lipid fluctuations near the phase transition.Abbreviations NMR Nuclear Magnetic Resonance - PC phosphatidylcholine - PE phosphatidylethanolamine - POPC Palmitoyloleoylphosphatidylcholine - POPE Palmitoyloleoylphosphatidylethanolamine - H_II Inverse hexagonal phase     

Characterisation of the effects of anthranilic and (indanyloxy) acetic acid derivatives on chloride transport in membrane vesicles.

The effects of the Cl- channel blockers, NPPB, IAA94/95 and a number of related compounds on 36Cl- transport in membrane vesicles from bovine kidney cortex and rabbit ileum mucosa brush borders have been studied. These vesicles have been previously shown to be enriched in Cl- channel and Cl-/anion cotransport activity, respectively. Chloride transport was assayed in both types of vesicles by measuring the uptake of 36Cl- in response to an outwardly-directed Cl- concentration gradient. In kidney microsomes, a large proportion of the observed 36Cl- uptake was mediated by an electrogenic uniport and could be substantially reduced by clamping the membrane potential at zero mV using K+ and valinomycin. Chloride uptake was inhibited by both NPPB and IAA94/95 with apparent IC50 values of around 10 microM under optimal conditions (i.e., 4 min uptake at 4 degrees C). Under other conditions (e.g., 10 min uptake at 25 degrees C), where uptake had reached a steady-state level, much higher concentrations of inhibitor were required to cause inhibition. Therefore, previous differences in the reported potency of these compounds may, in part, have been due to the conditions under which Cl- uptake was measured. In addition, both NPPB and, to a lesser extent, IAA94/95 were found to have other effects on the vesicles, in that, when added at a concentration of 100 microM, they induced a leakage of pre-accumulated 36Cl-. This was probably caused by either dissipation of membrane potential or damage to the vesicle membranes. The sulphonic acid derivatives of NPPB and IAA94/95 (NPPB-S and ISA94/95, respectively) blocked 36Cl- uptake with around the same potency as NPPB and IAA94/95, but did not cause any non-specific Cl- leakage, when added at concentrations up to 100 microM. Inhibition of 36Cl- uptake by all four compounds was almost completely reversible. However, when vesicles were incubated with the inhibitors in the presence of an outward Cl- concentration gradient, or if vesicles were freeze/thawed in the presence of the compounds, inhibition could be only partially reversed. In rabbit brush border membrane vesicles, 36Cl- uptake was not reduced when the vesicles were voltage clamped using valinomycin and K+, and was therefore probably mediated by Cl-/Cl- exchange. However, despite the lack of effect of valinomycin, 36Cl- uptake was inhibited by both NPPB (approx. 80% inhibition at 100 microM) and, to a lesser extent, by IAA94/95 (approx. 30% inhibition at 100 microM).(ABSTRACT TRUNCATED AT 400 WORDS)     

Social stress shortens lifespan in mice

Stress and low socioeconomic status in humans confer increased vulnerability to morbidity and mortality. However, this association is not mechanistically understood nor has its causation been explored in animal models thus far. Recently, cellular senescence has been suggested as a potential mechanism linking lifelong stress to age‐related diseases and shorter life expectancy in humans. Here, we established a causal role for lifelong social stress on shortening lifespan and increasing the risk of cardiovascular disease in mice. Specifically, we developed a lifelong chronic psychosocial stress model in which male mouse aggressive behavior is used to study the impact of negative social confrontations on healthspan and lifespan. C57BL/6J mice identified through unbiased cluster analysis for receiving high while exhibiting low aggression, or identified as subordinate based on an ethologic criterion, had lower median and maximal lifespan, and developed earlier onset of several organ pathologies in the presence of a cellular senescence signature. Critically, subordinate mice developed spontaneous early‐stage atherosclerotic lesions of the aortic sinuses characterized by significant immune cells infiltration and sporadic rupture and calcification, none of which was found in dominant subjects. In conclusion, we present here the first rodent model to study and mechanistically dissect the impact of chronic stress on lifespan and disease of aging. These data highlight a conserved role for social stress and low social status on shortening lifespan and increasing the risk of cardiovascular disease in mammals and identify a potential mechanistic link for this complex phenomenon.     

Supervillin (p205): A Novel Membrane-associated, F-Actin–binding Protein in the Villin/Gelsolin Superfamily

Actin-binding membrane proteins are involved in both adhesive interactions and motile processes. We report here the purification and initial characterization of p205, a 205-kD protein from bovine neutrophil plasma membranes that binds to the sides of actin filaments in blot overlays. p205 is a tightly bound peripheral membrane protein that cosediments with endogenous actin in sucrose gradients and immunoprecipitates. Amino acid sequences were obtained from SDS-PAGE–purified p205 and used to generate antipeptide antibodies, immunolocalization data, and cDNA sequence information. The intracellular localization of p205 in MDBK cells is a function of cell density and adherence state. In subconfluent cells, p205 is found in punctate spots along the plasma membrane and in the cytoplasm and nucleus; in adherent cells, p205 concentrates with E-cadherin at sites of lateral cell–cell contact. Upon EGTA-mediated cell dissociation, p205 is internalized with E-cadherin and F-actin as a component of adherens junctions “rings.” At later times, p205 is observed in cytoplasmic punctae. The high abundance of p205 in neutrophils and suspension-grown HeLa cells, which lack adherens junctions, further suggests that this protein may play multiple roles during cell growth, adhesion, and motility. Molecular cloning of p205 cDNA reveals a bipartite structure. The COOH terminus exhibits a striking similarity to villin and gelsolin, particularly in regions known to bind F-actin. The NH₂ terminus is novel, but contains four potential nuclear targeting signals. Because p205 is now the largest known member of the villin/gelsolin superfamily, we propose the name, “supervillin.” We suggest that supervillin may be involved in actin filament assembly at adherens junctions and that it may play additional roles in other cellular compartments.     

Heat shock proteins and heat adaptation of the whole organism

Moseley, Pope L. Heat shock proteins andheat adaptation of the whole organism. J. Appl.Physiol. 83(5): 1413-1417, 1997.Adaptation toheat may occur through acclimatization or thermotolerance; however, thelinkage of these phenomena is poorly understood. The importance of heatshock proteins (HSPs) in thermotolerance and differences in theiraccumulation in organisms adapted to the heat suggest a role for HSPsin acclimatization as well. The role of HSPs in heat adaptation of thewhole organism and the interrelationships among heat adaptation,endotoxin tolerance, and cytokine resistance through HSPs are reviewed.