Plasmon resonance spectroscopy: probing molecular interactions within membranes.

Surface plasmon resonance (SPR) has become a popular method for investigating biomolecular interactions. A new variant of this technique, coupled plasmon-waveguide resonance (CPWR) spectroscopy, allows the characterization of anisotropic biological membranes. Plasmon resonance can therefore be used to study the molecular events involved in a wide variety of membrane processes, including energy conversion and signal transduction.

Monoclonality of parathyroid tumors in chronic renal failure and in primary parathyroid hyperplasia.

The pathogeneses of parathyroid disease in patients with uremia and nonfamilial primary parathyroid hyperplasia are poorly understood. Because of multigland involvement, it has been assumed that these common diseases predominantly involve polyclonal (non-neoplastic) cellular proliferations, but an overall assessment of their clonality has not been done. We examined the clonality of these hyperplastic parathyroid tumors using X-chromosome inactivation analysis with the M27 beta (DXS255) DNA polymorphism and by searching for monoclonal allelic losses at M27 beta and at loci on chromosome band 11q13. Fully 7 of 11 informative hemodialysis patients (64%) with uremic refractory hyperparathyroidism harbored at least one monoclonal parathyroid tumor (with a minimum of 12 of their 19 available glands being monoclonal). Tumor monoclonality was demonstrable in 6 of 16 informative patients (38%) with primary parathyroid hyperplasia. Histopathologic categories of nodular versus generalized hyperplasia were not useful predictors of clonal status. These observations indicate that monoclonal parathyroid neoplasms are common in patients with uremic refractory hyperparathyroidism and also develop in a substantial group of patients with sporadic primary parathyroid hyperplasia, thereby changing our concept of the pathogenesis of these diseases. Neoplastic transformation of preexisting polyclonal hyperplasia, apparently due in large part to genes not yet implicated in parathyroid tumorigenesis and possibly including a novel X-chromosome tumor suppressor gene, is likely to play a central role in these disorders.

CONDENSED-MATTER SPECTROSCOPY SPECTRAL METHODS FOR STUDY OF THE G-PROTEIN-COUPLED RECEPTOR RHODOPSIN. II. MAGNETIC RESONANCE METHODS.

This article continues our review of spectroscopic studies of G-protein-coupled receptors. Magnetic resonance methods including electron paramagnetic resonance (EPR) and nuclear magnetic resonance (NMR) provide specific structural and dynamical data for the protein in conjunction with optical methods (vibrational, electronic spectroscopy) as discussed in the accompanying article. An additional advantage is the opportunity to explore the receptor proteins in the natural membrane lipid environment. Solid-state 2H and 13C NMR methods yield information about the both local structure and dynamics of the cofactor bound to the protein and its light induced changes. Complementary site-directed spin labeling studies monitor the structural alterations over larger distances and correspondingly longer time scales. A multi-scale reaction mechanism describes how local changes of the retinal cofactor unlock the receptor to initiate large-scale conformational changes of rhodopsin. Activation of the G-protein-coupled receptor involves an ensemble of conformational substates within the rhodopsin manifold that characterize the dynamically active receptor.

Resolution of independently titrating spectral components in the ultraviolet circular dichroism of subtilisin enzymes by matrix rank analysis.

The ultraviolet circular dichroism of di-isopropylphophoryl-subtilisins Carlsberg and Novo (EC 3.4.21.14) has been examined as a function of pH. The CD of these enzymes below 260 nm is invariant over the pH interval 4 to 12, below or above which spectral changes occur suggesting a transition to a random coil form. Above pH 8 contributions due to the ionization of tyrosyl residues appear in the CD above 260 nm as bands shifted to longer wavelengths. Three independently titratable components, obtained by matrix rank analysis, account for the observed CD spectral changes above 260 nm of Dip-subtilisin Carlsberg in the pH interval 8 to 12. By contrast, two components were derived for the Novo enzyme. The identities of the matrix rank components were surmised from their apparent pKa values. One component of both subtilisin enzymes corresponds to the CD of the "buried" or irreversibly titratable tyrosyl residues of the enzyme. The other matrix rank components correspond to the CD of the "exposed" or freely ionizable tyrosyl residues. These residues are optically active only in the ionized state. Two types of "exposed" tyrosyl residues, arising because of differing sensitivity to the ionization of the "partially buried" or abnormally titrating tyrosyl residues, are evident in Dip-subtilisin Carlsberg. A pH-induced local conformational change in this enzyme is proposed to account for this behavior. The "partially buried" tyrosyl residues of both subtilisins appear to be devoid of optical activity in either the tyrosyl or tyrosylate form.

Histidyl and tyrosyl residue ionization studies of subtilisin Novo.

The low field portion of the 360 MHz 1H nuclear magnetic resonance spectrum of phenylmethanesulfonyl-subtilisin Novo (EC 3.4.21.14) has been studied as a function of pH. Analysis of the pH-induced chemical shift changes occurring between 6 to 7 ppm revealed five classes of ionizable residues with pK values (uncorrected) of 10.3, 10.7, 10.7, 10.8, and 11.0. With a single exception the titration curves can be fit by assuming a simple proton ionization equilibrium. Four classes of low intensity broad resonances, assigned to the histidyl residues, are observed between 8 and 9 ppm. Uncorrected pK values of 5.4, 5.7, 6.0, and 6.4 were determined for the residues comprising each of these classes. The spectral data are consistent with protonation of one or more histidyl residues upon acid induced denaturation of the protein. These results are compared with those of analogues studies performed by the use of other techniques.

SPECTRAL METHODS FOR STUDY OF THE G-PROTEIN-COUPLED RECEPTOR RHODOPSIN. I. VIBRATIONAL AND ELECTRONIC SPECTROSCOPY.

Here we review the application of modern spectral methods for the study of G-protein-coupled receptors (GPCRs) using rhodopsin as a prototype. Because X-ray analysis gives us immobile snapshots of protein conformations, it is imperative to apply spectroscopic methods for elucidating their function: vibrational (Raman, FTIR), electronic (UV-visible absorption, fluorescence) spectroscopies, and magnetic resonance (electron paramagnetic resonance, EPR), and nuclear magnetic resonance, NMR). In the first of the two companion articles, we discuss the application of optical spectroscopy for studying rhodopsin in a membrane environment. Information is obtained regarding the time-ordered sequence of events in rhodopsin activation. Isomerization of the chromophore and deprotonation of the retinal Schiff base leads to a structural change of the protein involving the motion of helices H5 and H6 in a pH-dependent process. Information is obtained that is unavailable from X-ray crystallography, which can be combined with spectroscopic studies to achieve a more complete understanding of GPCR function.

Loss of caspase-3 sensitizes colon cancer cells to genotoxic stress via RIP1-dependent necrosis.

Caspase-3 is the best known executioner caspase in apoptosis. We generated caspase-3 knockout (C3KO) and knockdown human colorectal cancer cells, and found that they are unexpectedly sensitized to DNA-damaging agents including 5-fluorouracil (5-FU), etoposide, and camptothecin. C3KO xenograft tumors also displayed enhanced therapeutic response and cell death to 5-FU. C3KO cells showed intact apoptosis and activation of caspase-7 and -9, impaired processing of caspase-8, and induction of necrosis in response to DNA-damaging agents. This form of necrosis is associated with HMGB1 release and ROS production, and suppressed by genetic or pharmacological inhibition of RIP1, MLKL1, or caspase-8, but not inhibitors of pan-caspases or RIP3. 5-FU treatment led to the formation of a z-VAD-resistant pro-caspase-8/RIP1/FADD complex, which was strongly stabilized by caspase-3 KO. These data demonstrate a key role of caspase-3 in caspase-8 processing and suppression of DNA damage-induced necrosis, and provide a potentially novel way to chemosensitize cancer cells.

Both ischemic and pharmacological preconditioning decrease hepatic leukocyte/endothelial cell interactions.

BACKGROUND: Ischemic preconditioning has been shown to protect some tissues from ischemia/reperfusion (I/R) injury. Adenosine is believed to play an important role by attenuating leukocyte-endothelial cell adhesive interactions. Dipyridamole increases adenosine bioavailability. The purpose of this study was to evaluate the effects of mechanical (MPC) and pharmacological preconditioning (PPC) on leukocyte endothelial cell interaction in hepatic I/R injury. METHODS: C57BL6 mice were subjected to 30 min of ischemia to the left lobe of the liver. Groups tested at 30 min, 2, 5, 12, and 24 hr of reperfusion had 1) sham laparotomy (n = 10, 2) I/R (n = 25), 3) ischemic preconditioning with 5 min of ischemia and 10 min reperfusion before I/R (n = 25), and 4) (PPC) with dipyridamole (n = 25). Intravital microscopic examination was used to assess leukocyte/endothelial cell adhesion. Blood was drawn for leukocyte counts and liver function tests. RESULTS: A significant decrease in leukocyte rolling was observed at 30-min and 5-hr reperfusion intervals in the PPC and ischemic preconditioning groups compared with the I/R group. A significant decrease in leukocyte saltation was also observed in the PPC and MPC groups at 2, 5, and 12 hr of reperfusion when compared with the I/R group. aspartate aminotransferase was significantly decreased in the 5-hr preconditioning groups. There was not a significant decrease in the white blood cell count because of PPC or MPC vs. I/R CONCLUSIONS: Preconditioning decreases endothelial/ leukocyte interaction and reduces liver damage as measured by aspartate aminotransferase. These data prove that IPC and PPC provide some degree of hepatic protection in I/R injury.

Cloned human T cells synthesize Ia molecules and can function as antigen presenting cells.

TNP-specific proliferative cloned human T cell lines were investigated for their synthesis and cell surface expression of HLA-DR molecules and for their capacity to function as antigen presenting cells. Utilizing radioactive amino acid precursors for metabolic labeling, these studies demonstrated endogenous synthesis of HLA-DR molecules by cloned T cells, which by two-dimensional gel electrophoresis were similar to HLA-DR molecules expressed by B cells and monocytes. Moreover, when TNP-modified, the irradiated cloned T cells functioned very effectively to stimulate TNP-specific proliferation by cloned responders; when unmodified they were potent stimulators of allogeneic mixed leukocyte responses. Thus, for haptens covalently attached to cell membrane proteins and for allogeneic HLA antigens, Ia+ cloned T cells can function as effectively for antigen presentation and T cell activation as other Ia+ populations to which such properties have been ascribed.

Class II determinants recognized by TNP-specific cloned human T cell lines.

Cloned human T lymphocyte lines were generated against trinitrophenyl (TNP)-modified autologous cells from four different individuals. By examining the reactivity patterns of 58 of these T cell clones (TLC) on panels of HLA-typed antigen presenting cells and employing monoclonal antibodies (MoAb) for inhibition studies, we have demonstrated that TNP may be recognized in the context of different DR and DP associated determinants. We did not identify any TLC restricted by determinants associated uniquely with DQ. Determinants associated with the expression of DR1,2,4,5, and 7 as well as the supertypic specificities DRw52 (MT2) and DRw53 (MT3) functioned as restriction elements. In addition, at least two antigenically distinct regions of the DP4 molecule appeared to function in the presentation of TNP. One TNP-specific TLC recognized a determinant associated with the expression of DP4 while another recognized TNP in the context of a highly nonpolymorphic determinant on DP which was expressed on all stimulators tested. Monoclonal antibody blocking studies suggest that these two determinants lie on different portions of the DP molecule. These studies demonstrated that both polymorphic and relatively nonpolymorphic restriction determinants on DR and DP may function in the presentation of conventional antigen to autologous lymphocytes.

Kinetics of P-selectin expression in regional vascular beds after resuscitation of hemorrhagic shock: a clue to the mechanism of multiple system organ failure.

Leukocyte-endothelial cell interactions play an important role in mediating organ dysfunctions observed after hemorrhagic shock. P-selectin is the first endothelial cell adhesion molecule to be upregulated after an ischemic insult. The objective of this study was to define kinetics of P-selectin expression in different regional vascular beds of mice exposed to hemorrhagic shock. In-vivo P-selectin expressions were determined using dual radiolabeled monoclonal antibody technique in lungs, heart, liver, kidneys, intestinal mesentery, stomach, small bowel, and colon 0.5, 1, 2, 5, 10, and 24 h after resuscitation of 40 mmHg hemorrhagic shock. In another group, P-selectin expression was determined in same organs 5 h after resuscitation of 30 mmHg hemorrhagic shock. Hemorrhagic shock of 40 mmHg caused significant upregulation of P-selectin in lungs and liver at 30 min after resuscitation (P < 0.001). There was a second and more pronounced upregulation of P-selectin in lungs and liver at 5 h after resuscitation (P < 0.001). In heart, intestinal mesentery, stomach, small bowel, and colon, P-selectin was not upregulated until 5 h after resuscitation from 40 mmHg hemorrhagic shock (P < 0.001). While hemorrhagic shock of 40 mmHg did not cause P-selectin upregulation in kidneys, hemorrhage to 30 mmHg did elicit a significant increase at 5 h after resuscitation (P < 0.001). We conclude that P-selectin is upregulated after resuscitation of hemorrhagic shock in lungs, liver, heart, stomach, and intestines. P-selectin upregulation in kidneys only takes place after more severe hemorrhagic shock.

Preoperative vein mapping for coronary artery bypass operations.

High-resolution, real-time ultrasonographic imaging of the greater saphenous veins was performed preoperatively in 100 patients undergoing coronary artery bypass grafting. Vein diameters were measured by ultrasound at four locations in the leg, and the course of the vein in the leg was marked on the overlying skin. Veins removed at operation were measured at the same locations at initial dissection and after vein preparation. The mapped and in situ vein diameters correlated closely, whereas the distended vein diameter was approximately 1.5 mm larger. When greater saphenous veins were absent or diseased, lesser saphenous veins were mapped. No differences in measurements were demonstrated for a variety of patient and operator variables. Major branches or duplications were predicted correctly in 11 patients and venous disease in 13 patients. Mapping influenced the surgeon's choice of the venectomy site in 13 patients. Vein mapping is a simple, accurate, and noninvasive method of imaging the saphenous vein preoperatively. It is useful in demonstrating areas of venous anomalies and disease, and predicts the course of the vein in the leg.

Does a cognitive map guide choices in the radial-arm maze?

Fifteen rats performed in a standard radial-arm maze task (Experiment 1) and in a modified task with a set of forced choices and a 15-min retention interval prior to completion of the maze (Experiment 2). In addition to the standard measure of choice in the radial-arm maze, orientation toward arms was measured and considered to constitute go-no-go "microchoice" decisions. Rats investigated but rejected many arms. A model of choice was developed in which it was assumed that choice decisions about arms were made independently and that microchoices were not selectively guided toward baited arms. The model performed nearly as well as the rats. These results place important limitations on the theory that choice behavior in the radial-arm maze is guided by a cognitive map.

Element and compound matching-to-sample performance in pigeons: the roles of information load and training history.

Pigeons were trained to match color and line orientation element or compound samples in a symbolic matching-to-sample task. In subsequent test sessions with element and compound samples, there was an initial superiority of element matching for the element-trained group and of compound matching for the compound-trained group. This difference persisted over the course of 100 test sessions for the element-trained group, whereas element- and compound-matching accuracy converged for the compound-trained group. In a second experiment, in which sample duration was manipulated, element-matching accuracy was superior to compound-matching accuracy for both groups. Thus, element-matching accuracy was superior to compound-matching accuracy under conditions that rule out generalization decrement and training history as explanations. The data are interpreted as supporting the view that the dimensions of visual compound stimuli compete for a limited cognitive resource.

Control of choice by the spatial configuration of goals.

Rats gathered pellets from the tops of 15.5-cm-tall poles. In a matrix of poles, bait was located on the tops of poles arranged in either a square (Experiment 1) or linear (Experiment 2) configuration. The specific locations of baited poles varied unpredictably from trial to trial. The data show that the rats' choices were controlled by the spatial configuration of baited locations. This indicates that the rats represented the geometric pattern formed by the locations of food.

Spatial pattern learning in rats: control by an iterative pattern.

Rats searched in a matrix of vertical poles for food hidden on top of the poles. The only information available about the location of the food was the consistent spatial pattern of the baited poles, which was a checkerboard. This spatial pattern of hidden-food locations came to control the choices of poles made by the rats. The experiments ruled out the possibility that this control can be explained by the acquisition of simple response tendencies to move from pole to pole. Instead, this behavioral control of choices was attributed to the development of a representation of the checkerboard pattern of baited locations. Spatial pattern learning may have mechanisms in common with other forms of pattern learning.

Flexible memory processing by rats: use of prospective and retrospective information in the radial maze.

Four experiments investigated the content of the memory used by rats in mediating retention intervals interpolated during performance in a 12-arm radial maze. The delay occurred following either the 2nd, 4th, 6th, 8th, or 10th choice. A 15-min delay had the greatest disruptive effect when interpolated in the middle of the choice sequence and less of an effect when it occurred either earlier or later. This pattern of results was obtained when either a free- or forced-choice procedure was used prior to the delay and regardless of whether postdelay testing consisted of completion of the maze or two-alternative forced-choice tests. Assuming that the disruptive effect of a delay is a function of memory load, this implies that the rats used information about previously visited arms (retrospective memory) following an earlier interpolated delay but information about anticipated choices (prospective memory) following a delay interpolated late in the choice sequence. There appeared to be a recency effect only in the early and middle delay conditions. This provides converging evidence for the dual-code hypothesis. No evidence for prospective memory was obtained following a 60-min delay.

Spatial guidance of choice behavior in the radial-arm maze.

In 6 experiments, the performance of male rats in a 12-arm radial maze was examined. The focus of study was the extent to which the spatial location of individual baited maze arms was determined before the rat was exposed to the extramaze visual cues corresponding to the arm, and thereby guided the rat toward the location of baited arms. Such spatial guidance of choice behavior implies a spatially organized cognitive representation of maze arms (i.e., a cognitive map). A higher level of spatial guidance was found when visual access to extramaze cues was restricted than when it was unrestricted. There was no evidence of a difference between the level of spatial guidance in the context of working memory performance and reference memory performance. Some evidence that intramaze cues contributed to microchoice guidance was found. However, spatial guidance, under at least some conditions, is best explained in terms of cognitive mapping.

Corneal lenticular wrinkling after automated lamellar keratoplasty.

PURPOSE: To report complications of automated lamellar keratoplasty in two eyes of two patients. METHODS: Case reports. Two eyes of two patients underwent automated lamellar keratoplasty for myopia. Both patients complained of visual distortion and glare in the postoperative eye. RESULTS: The postoperative eye of both patients showed evidence of wrinkling of the corneal lenticule accompanied by irregular astigmatism. Patient 1 showed persistent lenticular wrinkling and corneal scarring 2.5 years later. Patient 2 showed evidence of interface scar and overcorrection. CONCLUSION: The use of a microkeratome can be complicated by lenticular displacement and wrinkling, resulting in visual aberration for the patient.

Membrane lipid influences on the energetics of the metarhodopsin I and metarhodopsin II conformational states of rhodopsin probed by flash photolysis.

We have investigated the relationship between rhodopsin photochemical function and the retinal rod outer segment (ROS) disk membrane lipid composition using flash photolysis techniques. Bovine rhodopsin was combined with various phospholipids to form recombinant membrane vesicles, in which the lipid acyl chain composition was maintained at that of egg phosphatidylcholine (PC), while the nature of the headgroups was varied. The ratio of metarhodopsin II (MII)/metarhodopsin I (MI) in these recombinants produced by an actinic flash was investigated as a function of pH, and compared with the photochemical activity observed for rhodopsin in native ROS membranes and dimyristoylphosphatidylcholine recombinants. In recombinants made with lipids derived from egg PC, as well as in native ROS membranes, MI and MII were found to be present in a pH-dependent, acid-base equilibrium on the millisecond timescale. The recombinants made with phospholipids containing unsaturated acyl chains were capable of full native-like MII production, but each demonstrated a titration curve with a different pK. In addition, some of the recombinants exhibited apparent deviations from the Henderson-Hasselbalch curve shape. The presence of either phosphatidylethanolamine (PE) or phosphatidylserine (PS) headgroups appeared to increase the amount of MII produced. This may result from alteration of the curvature free energy, in the case of PE, and from the influence of the membrane surface potential in the case of PS. An investigation of the effects of temperature on the MI-MII transition in native ROS membranes and the recombinants was also carried out.(ABSTRACT TRUNCATED AT 250 WORDS)