Transmission Raman Measurements Using a Spatial Heterodyne Raman Spectrometer (SHRS).

A spatial heterodyne Raman spectrometer (SHRS) was used to measure transmission Raman spectra of highly scattering compounds. Transmission Raman spectral intensities of ibuprofen were only 2.4 times lower in intensity than backscatter Raman spectra. The throughput was about eight times higher than an f/1.8 dispersive spectrometer, and the width of the area viewed was found to be seven to nine times higher, using 50.8 mm and 250 mm focal length collection lenses. However, the signal-to-noise (S/N) ratio was two times lower for the SHRS than the f/1.8 dispersive spectrometer, apparently due to high levels of stray light.

Improving Spectral Results Using Row-by-Row Fourier Transform of Spatial Heterodyne Raman Spectrometer Interferogram.

This work describes a method of applying the Fourier transform to the two-dimensional Fizeau fringe patterns generated by the spatial heterodyne Raman spectrometer (SHRS), a dispersive interferometer, to correct the effects of certain types of optical alignment errors. In the SHRS, certain types of optical misalignments result in wavelength-dependent and wavelength-independent rotations of the fringe pattern on the detector. We describe here a simple correction technique that can be used in post-processing, by applying the Fourier transform in a row-by-row manner. This allows the user to be more forgiving of fringe alignment and allows for a reduction in the mechanical complexity of the SHRS.

Developmental regulation of a novel outwardly rectifying mechanosensitive anion channel in Caenorhabditis elegans.

The nematode Caenorhabditis elegans offers unique experimental advantages for defining the molecular basis of anion channel function and regulation. However, the relative inaccessibility of somatic cells in adult animals greatly limits direct electrophysiological studies of channel activity. We developed methods to routinely isolate and patch clamp C. elegans embryo cells and oocytes and to culture and patch clamp neurons and muscle cells. Dissociated embryonic cells express a robust outwardly rectifying anion current that is activated by membrane stretch and depolarization. This current, termed I(Cl,mec), is inhibited by anion and mechanosensitive channel inhibitors. I(Cl,mec) has broad anion selectivity and the channel has a unitary conductance of 5-7 picosiemens. I(Cl,mec) is not detectable in whole-cell or isolated patch recordings from oocytes, cultured muscle cells, and cultured neurons but is expressed in single cell and later embryos. Channel density is high, and the current is observed in >80% of membrane patches. Macroscopic currents of 40-120 pA at +100 mV are typically observed in inside-out membrane patches formed using low resistance patch pipettes. Isolated membrane patches of early embryonic cells therefore contain 60-200 I(Cl,mec) channels. The apparent activation of I(Cl,mec) shortly after fertilization and its down-regulation in terminally differentiated cells suggests that the channel may play important roles in embryogenesis and/or cytokinesis.

Validation of a new algorithm for the BPM-100 electronic oscillometric office blood pressure monitor.

BACKGROUND: To test the accuracy of a new algorithm for the BPM-100, an automated oscillometric blood pressure (BP) monitor, using stored data from an independently conducted validation trial comparing the BPM-100(Beta) with a mercury sphygmomanometer. DESIGN: Raw pulse wave and cuff pressure data were stored electronically using embedded software in the BPM-100(Beta), during the validation trial. The 391 sets of measurements were separated objectively into two subsets. A subset of 136 measurements was used to develop a new algorithm to enhance the accuracy of the device when reading higher systolic pressures. The larger subset of 255 measurements (three readings for 85 subjects) was used as test data to validate the accuracy of the new algorithm. METHODS: Differences between the new algorithm BPM-100 and the reference (mean of two observers) were determined and expressed as the mean difference +/- SD, plus the percentage of measurements within 5, 10, and 15 mmHg. RESULTS: The mean difference between the BPM-100 and reference systolic BP was -0.16 +/- 5.13 mmHg, with 73.7% < or = 5 mmHg, 94.9% < or = 10 mmHg and 98.8% < or = 15 mmHg. The mean difference between the BPM-100 and reference diastolic BP was -1.41 +/- 4.67 mmHg, with 78.4% < or = 5 mmHg, 92.5% < or = 10 mmHg, and 99.2% < or = 15 mmHg. These data improve upon that of the BPM-100(Beta) and pass the AAMI standard, and 'A' grade BHS protocol. CONCLUSION: This study illustrates a new method for developing and testing a change in an algorithm for an oscillometric BP monitor utilizing collected and stored electronic data and demonstrates that the new algorithm meets the AAMI standard and BHS protocol.

Regulation of I(Cl,swell) in neuroblastoma cells by G protein signaling pathways.

Guanosine 5'-O-(3-thiotriphosphate) (GTPgammaS) activated the I(Cl,swell) anion channel in N1E115 neuroblastoma cells in a swelling-independent manner. GTPgammaS-induced current was unaffected by ATP removal and broadly selective tyrosine kinase inhibitors, demonstrating that phosphorylation events do not regulate G protein-dependent channel activation. Pertussis toxin had no effect on GTPgammaS-induced current. However, cholera toxin inhibited the current approximately 70%. Exposure of cells to 8-bromoadenosine 3',5'-cyclic monophosphate did not mimic the effect of cholera toxin, and its inhibitory action was not prevented by treatment of cells with an inhibitor of adenylyl cyclase. These results demonstrate that GTPgammaS does not act through Galpha(i/o) GTPases and that Galpha(s)/Gbetagamma G proteins inhibit the channel and/or channel regulatory mechanisms through cAMP-independent mechanisms. Swelling-induced activation of I(Cl,swell) was stimulated two- to threefold by GTPgammaS and inhibited by 10 mM guanosine 5'-O-(2-thiodiphosphate). The Rho GTPase inhibitor Clostridium difficile toxin B inhibited both GTPgammaS- and swelling-induced activation of I(Cl,swell). Taken together, these findings indicate that Rho GTPase signaling pathways regulate the I(Cl,swell) channel via phosphorylation-independent mechanisms.

CLH-3, a ClC-2 anion channel ortholog activated during meiotic maturation in C. elegans oocytes.

BACKGROUND: ClC anion channels are ubiquitous and have been identified in organisms as diverse as bacteria and humans. Despite their widespread expression and likely physiological importance, the function and regulation of most ClCs are obscure. The nematode Caenorhabditis elegans offers significant experimental advantages for defining ClC biology. These advantages include a fully sequenced genome, cellular and molecular manipulability, and genetic tractability. RESULTS: We show by patch clamp electrophysiology that C. elegans oocytes express a hyperpolarization- and swelling-activated Cl(-) current with biophysical characteristics strongly resembling those of mammalian ClC-2. Double-stranded RNA-mediated gene interference (RNAi) and single-oocyte RT-PCR demonstrated that the channel is encoded by clh-3, one of six C. elegans ClC genes. CLH-3 is inactive in immature oocytes but can be triggered by cell swelling. However, CLH-3 plays no apparent role in oocyte volume homeostasis. The physiological signal for channel activation is the induction of oocyte meiotic maturation. During meiotic maturation, the contractile activity of gonadal sheath cells, which surround oocytes and are coupled to them via gap junctions, increases dramatically. These ovulatory sheath cell contractions are initiated prematurely in animals in which CLH-3 expression is disrupted by RNAi. CONCLUSIONS: The inwardly rectifying Cl(-) current in C. elegans oocytes is due to the activity of a ClC channel encoded by clh-3. Functional and structural similarities suggest that CLH-3 and mammalian ClC-2 are orthologs. CLH-3 is activated during oocyte meiotic maturation and functions in part to modulate ovulatory contractions of gap junction-coupled gonadal sheath cells.

Dependence of KCC2 K-Cl cotransporter activity on a conserved carboxy terminus tyrosine residue.

K-Cl cotransporters (KCC) play fundamental roles in ionic and osmotic homeostasis. To date, four mammalian KCC genes have been identified. KCC2 is expressed exclusively in neurons. Injection of Xenopus oocytes with KCC2 cRNA induced a 20-fold increase in Cl(-)-dependent, furosemide-sensitive K(+) uptake. Oocyte swelling increased KCC2 activity 2-3 fold. A canonical tyrosine phosphorylation site is located in the carboxy termini of KCC2 (R1081-Y1087) and KCC4, but not in other KCC isoforms. Pharmacological studies, however, revealed no regulatory role for phosphorylation of KCC2 tyrosine residues. Replacement of Y1087 with aspartate or arginine dramatically reduced K(+) uptake under isotonic and hypotonic conditions. Normal or near-normal cotransporter activity was observed when Y1087 was mutated to phenylalanine, alanine, or isoleucine. A tyrosine residue equivalent to Y1087 is conserved in all identified KCCs from nematodes to humans. Mutation of the Y1087 congener in KCC1 to aspartate also dramatically inhibited cotransporter activity. Taken together, these results suggest that replacement of Y1087 and its congeners with charged residues disrupts the conformational state of the carboxy terminus. We postulate that the carboxy terminus plays an essential role in maintaining the functional conformation of KCC cotransporters and/or is involved in essential regulatory protein-protein interactions.

Effect of social housing condition on heat shock protein (HSP) expression in the Shionogi mouse mammary carcinoma (SC115).

Our previous studies have shown that social housing conditions can significantly alter the growth rate of the Shionogi mouse mammary carcinoma (SC115). The present study extended our investigations to the molecular level by examining stressor effects on the expression of a group of stress-responsive proteins, the heat shock proteins (HSPs). We hypothesized that HSP expression in SC115 cells may be altered by (a) different social housing conditions in vivo and (b) steroid hormone and growth factor exposure in vitro. Mice were reared in groups (G) or as individuals (I). Immediately following tumor cell injection, mice were rehoused from group to individual (GI), from individual to group (IG), or they remained in groups (GG). Tumor tissue was resected at 0.8 g or 3.0 g, as evidence suggests that tumor size affects HSP expression, which in turn affects proliferation. The data demonstrate that expression of HSP25, 70, and 90 was increased in tumors from mice in the IG compared to GG and GI mice, at both tumor weights examined. In addition, in IG mice, HSP90 expression was greater in 0.8 g compared to 3.0 g tumors. Under controlled culture conditions, hormones known to stimulate SC115 growth both in vivo and in vitro altered HSP expression. Physiological levels of dihydrotestosterone (DHT) and pharmacological levels of hydrocortisone (HC) upregulated expression of HSP25, whereas physiological levels of beta-estradiol (E2) upregulated expression of HSP90. These data are the first to demonstrate that a psychosocial stressor, a change in social housing condition, can induce differential HSP expression. Further, these data show that hormones that regulate SC115 tumor growth, also alter HSP expression.

Psychosocial stressors and mammary tumor growth: an animal model.

Stressful life events and the ability to cope with stress may play a role in the progression of breast cancer; however, the complex relationship between stressors and tumor growth is difficult to investigate in humans. Our studies have utilized the androgen-responsive Shionogi mouse mammary carcinoma (AR SC115) in male mice to investigate the effects of social housing condition on tumor growth rates and responses to chemotherapy. We demonstrate that, depending on social housing condition, mammary tumor growth and response to chemotherapy can both increase and decrease. We have examined the possible role(s) of 1) psychosocial variables, 2) testosterone and corticosterone, hormones altered by stress and known to stimulate SC115 cells in vivo and in vitro, 3) NK cells, one of the body's first lines of defense against tumor cells, 4) stress proteins, in mediating the differential tumor growth rates observed in our model. This review discusses the investigations we have undertaken to elucidate the mechanisms through which a psychosocial stressor, social housing condition, can alter tumor growth rate.

Restoration of use of paralyzed limb muscles using sensory nerve signals for state control of FES-assisted walking.

A real-time functional electrical stimulation (FES) state controller was designed that utilized sensory nerve cuff signals from the cat forelimb to control the timing of stimulation of the Palmaris Longus (PalL) muscle during walking on the treadmill. Sensory nerve signals from the median and superficial radial nerves provided accurate, reliable feedback related to foot contact and lift-off which, when analyzed with single threshold Schmitt triggers, produced valuable state information about the step cycle. The study involved three experiments: prediction of the timing of muscle activity in an open-loop configuration with no stimulation, prediction of the timing of muscle activity in a closed-loop configuration that included stimulation of the muscle over natural PaIL electromyogram (EMG), and temporary paralysis of selected forelimb muscles coupled with the use of the state controller to stimulate the PalL in order to return partial support function to the anesthetized limb. The FES state controller was tested in a variety of walking conditions, including different treadmill speeds and slopes. The results obtained in these experiments demonstrate that nerve cuff signals can provide a useful source of feedback to FES systems for control of limb function.

Gait phase information provided by sensory nerve activity during walking: applicability as state controller feedback for FES.

In this study, we extracted gait-phase information from natural sensory nerve signals of primarily cutaneous origin recorded in the forelimbs of cats during walking on a motorized treadmill. Nerve signals were recorded in seven cats using nerve cuff or patch electrodes chronically implanted on the median, ulnar, and/or radial nerves. Features in the electroneurograms that were related to paw contact and lift-off were extracted by threshold detection. For four cats, a state controller model used information from two nerves (either median and radial, or ulnar and radial) to predict the timing of palmaris longus activity during walking. When fixed thresholds were used across a variety of walking conditions, the model predicted the timing of EMG activity with a high degree of accuracy (average error = 7.8%, standard deviation = 3.0%, n = 14). When thresholds were optimized for each condition, predictions were further improved (average error = 5.5%, standard deviation = 2.3%, n = 14). The overall accuracy with which EMG timing information could be predicted using signals from two cutaneous nerves for two constant walking speeds and three treadmill inclinations for four cats suggests that natural sensory signals may be implemented as a reliable source of feedback for closed-loop control of functional electrical stimulation (FES).

ATP dependence of the ICl,swell channel varies with rate of cell swelling. Evidence for two modes of channel activation.

Swelling-induced activation of the outwardly rectifying anion current, ICl, swell, is modulated by intracellular ATP. The mechanisms by which ATP controls channel activation, however, are unknown. Whole cell patch clamp was employed to begin addressing this issue. Endogenous ATP production was inhibited by dialyzing N1E115 neuroblastoma cells for 4-5 min with solutions containing (microM): 40 oligomycin, 5 iodoacetate, and 20 rotenone. The effect of ATP on current activation was observed in the absence of intracellular Mg2+, in cells exposed to extracellular metabolic inhibitors for 25-35 min followed by intracellular dialysis with oligomycin, iodoacetate, and rotenone, after substitution of ATP with the nonhydrolyzable analogue AMP-PNP, and in the presence of AMP-PNP and alkaline phosphatase to dephosphorylate intracellular proteins. These results demonstrate that the ATP dependence of the channel requires ATP binding rather than hydrolysis and/or phosphorylation reactions. When cells were swollen at 15-55%/min in the absence of intracellular ATP, current activation was slow (0.3-0.8 pA/pF per min). ATP concentration increased the rate of current activation up to maximal values of 4-6 pA/pF per min, but had no effect on the sensitivity of the channel to cell swelling. Rate of current activation was a saturable, hyperbolic function of ATP concentration. The EC50 for ATP varied inversely with the rate of cell swelling. Activation of current was rapid (4-6 pA/pF per min) in the absence of ATP when cells were swollen at rates >/=65%/min. Intracellular ATP concentration had no effect on current activation induced by high rates of swelling. Current activation was transient when endogenous ATP was dialyzed out of the cytoplasm of cells swollen at 15%/min. Rundown of the current was reversed by increasing the rate of swelling to 65%/min. These results indicate that the channel and/or associated regulatory proteins are capable of sensing the rate of cell volume increase. We suggest that channel activation occurs via ATP-dependent and -independent mechanisms. Increasing the rate of cell swelling appears to increase the proportion of channels activating via the ATP-independent pathway. These findings have important physiological implications for understanding ICl, swell regulation, the mechanisms by which cells sense volume changes, and volume homeostasis under conditions where cell metabolism is compromised.

Recombinant pICln forms highly cation-selective channels when reconstituted into artificial and biological membranes.

pICln has been proposed to be the swelling-activated anion channel responsible for ICl, swell, or a channel regulator. We tested the anion channel hypothesis by reconstituting recombinant pICln into artificial and biological membranes. Single channels were observed when pICln was reconstituted into planar lipid bilayers. In the presence of symmetrical 300 mM KCl, the channels had a high open probability and a slope conductance of 48 pS, and were outwardly rectifying. Reduction of trans KCl to 50 mM shifted the reversal potential by -31.2 +/- 0.06 mV, demonstrating that the channel is at least seven times more selective for cations than for anions. Consistent with this finding, channel conductance was unaffected by substitution of Cl- with glutamate, but was undetectable when K+ was replaced by N-methyl-D-glucamine. Reconstitution of pICln into liposomes increased 86Rb+ uptake by three- to fourfold, but had no effect on 36Cl- uptake. Phosphorylation of pICln with casein kinase II or mutation of G54, G56, and G58 to alanine decreased channel open probability and 86Rb+ uptake. When added to the external medium bathing Sf9 cells, pICln inserted into the plasma membrane and increased cell cation permeability. Taken together, these observations demonstrate that channel activity is due to pICln and not minor contaminant proteins. However, these findings do not support the hypothesis that pICln is the anion-selective ICl, swell channel. The observed cation channel activity may reflect an as yet to be defined physiological function of pICln, or may be a consequence of in vitro reconstitution of purified, recombinant protein.

Functional demonstration of Na+-K+-2Cl- cotransporter activity in isolated, polarized choroid plexus cells.

The function of the apical Na+-K+-2Cl- cotransporter in mammalian choroid plexus (CP) is uncertain and controversial. To investigate cotransporter function, we developed a novel dissociated rat CP cell preparation in which single, isolated cells maintain normal polarized morphology. Immunofluorescence demonstrated that in isolated cells the Na+-K+-ATPase, Na+-K+-2Cl- cotransporter, and aquaporin 1 water channel remained localized to the brush border, whereas the Cl-/HCO-3 (anion) exchanger type 2 was confined to the basolateral membrane. We utilized video-enhanced microscopy and cell volume measurement techniques to investigate cotransporter function. Application of 100 microM bumetanide caused CP cells to shrink rapidly. Elevation of extracellular K+ from 3 to 6 or 25 mM caused CP cells to swell 18 and 33%, respectively. Swelling was blocked completely by Na+ removal or by addition of 100 microM bumetanide. Exposure of CP cells to 5 mM BaCl2 induced rapid swelling that was inhibited by 100 microM bumetanide. We conclude that the CP cotransporter is constitutively active and propose that it functions in series with Ba2+-sensitive K+ channels to reabsorb K+ from cerebrospinal fluid to blood.

Characterization of pI(Cln) binding proteins: identification of p17 and assessment of the role of acidic domains in mediating protein-protein interactions.

pICln is a ubiquitous and abundant 27 kDa soluble protein that is localized primarily to the cytoplasm. The protein has been proposed to be a swelling-activated anion channel or a channel regulator. Recent studies, however, have cast significant doubt on these hypotheses, and the function of pI(Cln) therefore remains unknown. To further characterize the physiological role of pI(Cln), we have begun to identify the proteins that bind to it and the amino acid domains that mediate pICln protein-protein interactions. Using affinity assays and immunoprecipitation we have identified three proteins in C6 glioma cells with molecular masses of 17 kDa, 29 kDa and 72 kDa that bind selectively to pI(Cln). Microsequencing revealed that p17 is the non-muscle isoform of the alkali myosin light chain. pI(Cln) contains three acidic amino acid domains termed AD1, AD2 and AD3. Mutation of AD1 and/or AD2 had no effect on p17, p29 and p72 binding. However, binding of p72 was lost when four acidic amino acid residues were mutated in AD3, which is located at the carboxy terminus. A truncation peptide containing the last 29 amino acids of pI(Cln) was able to bind p72 normally. These results indicate that the carboxy terminus is necessary for p72-pI(Cln) interaction. Based on these and other findings, we propose that pI(Cln) is a protein responsible for regulating the structure and function of the cytoskeleton, and/or a protein involved in mediating interactions between components of intracellular signal transduction pathways.

Intracellular ionic strength regulates the volume sensitivity of a swelling-activated anion channel.

Cell swelling activates an outwardly rectifying anion channel termed VSOAC (volume-sensitive organic osmolyte/anion channel). Regulation of VSOAC by intracellular electrolytes was characterized in Chinese hamster ovary cells by whole cell patch clamp. Elevation of intracellular CsCl concentration from 40 to 180 mM resulted in a concentration-dependent decrease in channel activation. Activation of VSOAC was insensitive to the salt gradient across the plasma membrane, the intracellular concentration of specific anions or cations, and the total intracellular concentration of cations, anions, or electrolytes. Comparison of cells dialyzed with either CsCl or Na2SO4 solutions demonstrated directly that VSOAC activation is modulated by intracellular ionic strength (microi). The relative cell volume at which VSOAC current activation was triggered, termed the channel volume set point, decreased with decreasing ionic strength. At microi = 0.04, VSOAC activation occurred spontaneously in shrunken cells. The rate of VSOAC activation was nearly 50-fold higher in cells with microi = 0.04 vs. those with microi = 0.18. We propose that microi modulates the volume sensor responsible for channel activation.

Characterization of pICln phosphorylation state and a pICln-associated protein kinase.

pICln is a ubiquitous cellular protein that has been proposed to be a volume-sensitive Cl- channel or a channel regulator. Detailed biochemical, cellular and molecular characterization of pICln is required to understand its function. Our goal in the present investigation was to define further the biochemical properties of pICln and the proteins that associate with it. Immunoprecipitation of pICln from 32P-orthophosphoric acid-labeled C6 glioma cells revealed that the protein is phosphorylated constitutively, primarily on serine residues. Protein kinase activity was detected in pICln immunoprecipitates, revealing that a constitutively active protein kinase co-precipitates with pICln. A specific association between pICln and a protein kinase was also observed in affinity assays using a recombinant GST-pICln fusion protein. The pICln-associated kinase displayed broad substrate specificity and was inhibited in a concentration-dependent manner by heparin, zinc and 5,6-dichloro-1-beta-D-ribofuranosylbenose (DRB). These characteristics resembled those of casein kinase I and II. The pICln-associated kinase was not recognized, however, by antibodies against these two enzymes. Association of the kinase with pICln was disrupted by increasing concentrations of NaCl in the washing buffer, suggesting that electrostatic interactions are involved in kinase binding. Mutagenesis experiments corroborated this observation. Truncation of pICln demonstrated that two highly charged clusters of acidic amino acid residues are both necessary and sufficient for kinase binding. Phosphopeptide mapping demonstrated that pICln contains at least two phosphorylated serine residues that are located on trypsin cleavage fragments rich in acidic amino acid residues. We propose that the kinase or a kinase binding protein binds to acidic amino acids located between D101 and Y156 and phosphorylates nearby serine residues.

Effect of cell swelling on membrane and cytoplasmic distribution of pICln.

pICln is found ubiquitously in mammalian cells and is postulated to play a critical role in cell volume regulation. Mutagenesis studies led to the proposal that pICln is a swelling-activated anion channel. However, recent studies in Madin-Darby canine kidney cells and endothelial cells have shown that the protein is localized primarily to the cytoplasm. It has therefore been postulated that activation involves reversible translocation of pICln from the cytoplasm and insertion into the plasma membrane. We tested this hypothesis using several different approaches. Fractionation of C6 glioma cells into plasma membrane- and cytoplasm-containing fractions demonstrated that approximately 90% of the recovered pICln was confined to the cytosol. Swelling had no effect on the relative amount of protein present in the plasma membrane fraction. Immunofluorescence microscopy revealed that pICln is localized primarily, if not exclusively, to the cytoplasm of swollen and nonswollen cells. Similarly, transfection of cells with a green fluorescent protein-labeled pICln construct failed to reveal any membrane localization of the protein. These findings do not support the hypothesis that pICln is a volume regulatory anion channel activated by swelling-induced membrane insertion.