Osmolyte-driven contraction of a random coil protein

The Stokes radius characteristics of reduced and carboxamidated ribonuclease A (RCAM RNase) were determined for transfer of this "random coil" protein from water to 1 M concentrations of the naturally occurring protecting osmolytes trimethylamine N-oxide, sarcosine, sucrose, and proline and the nonprotecting osmolyte urea. The denatured ensemble of RCAM RNase expands in urea and contracts in protecting osmolytes to extents proportional to the transfer Gibbs energy of the protein from water to osmolyte. This proportionality suggests that the sum of the transfer Gibbs energies of individual parts of the protein is responsible for the dimensional changes in the denatured ensemble. The dominant term in the transfer Gibbs energy of RCAM RNase from water to protecting osmolytes is the unfavorable interaction of the osmolyte with the peptide backbone, whereas the favorable interaction of urea with the backbone dominates in RCAM RNase transfer to urea. The side chains collectively favor transfer to the osmolytes, with some protecting osmolytes solubilizing hydrophobic side chains as well as urea does, a result suggesting there is nothing special about the ability of urea to solubilize hydrophobic groups. Protecting osmolytes stabilize proteins by raising the chemical potential of the denatured ensemble, and the uniform thermodynamic force acting on the peptide backbone causes the collateral effect of contracting the denatured ensemble. The contraction decreases the conformational entropy of the denatured state while increasing the density of hydrophobic groups, two effects that also contribute to the ability of protecting osmolytes to force proteins to fold.     

The effects of platelet-derived growth factor-BB on healing of the rabbit medial collateral ligament. An in vivo study

Cigarette smoking poses significant risk to mother and infant during pregnancy and the postpartum period. Recruitment of pregnant smokers to intervention studies has often been reactive and has excluded certain subgroups of women, such as those who have recently quit smoking. In this study, we examined smoking patterns among a proactively recruited sample of women presenting to six urban community maternity clinics. The current report describes the patterns of smoking in this population of ethnoculturally diverse low-income urban pregnant women and examines differences across subgroups. The majority of the total sample in the current study reported that they had never smoked. Of the total, 30% reported having "ever" smoked and 16% were current smokers. Of the group of "ever" smokers, 18% quit greater than 12 months before pregnancy, 5% quit 0-12 months before pregnancy and 23% quit during this pregnancy. On the average, women who quit during pregnancy did so about 5 weeks after diagnosis. Of those women who continued to smoke during pregnancy, the average number of cigarettes smoked per day was 10 +/- 8. Differences were found in smoking patterns across the ethnocultural subgroups. Recruitment represents the first and one of the most important phases in intervening with pregnant women. Inclusion of both current smokers and recent self-quitters takes the fullest advantage of the window of opportunity to help women quit smoking and remain cigarette free for good.     

Patterns of faciodental sexual dimorphism in Hispanopithecus

The lipid bilayer technique was used to examine the effects of the ATP-sensitive K+ channel inhibitor (glibenclamide) and openers (diazoxide, minoxidil and cromakalim) and Cl- channel activators (GABA and diazepam) on two types of chloride channels in the sarcoplasmic reticulum (SR) from rabbit skeletal muscle. Neither diazepam at 100 microM nor GABA at 150 microM had any significant effect on the conductance and kinetics of the 75 pS small chloride (SCl) channel. Unlike the 150 pS channel, the SCl channel is sensitive to cytoplasmic glibenclamide with Ki approximately 30 microM. Glibenclamide induced reversible decline in the values of current (maximal current amplitude, Imax and average mean current, I') and kinetic parameters (frequency of opening Fo, probability of the channel being open Po and mean open time, To, of the SCl channel. Glibenclamide increased mean closed time, Tc, and was a more potent blocker from the cytoplasmic side (cis) than from the luminal side (trans) of the channel. Diazoxide increased I', Po, and To in the absence of ATP and Mg2+ but it had no effect on Imax and also failed to activate or remove the glibenclamide- and ATP-induced inhibition of the SCl channel. Minoxidil induced a transient increase in I' followed by an inhibition of Imax, whereas cromakalim reduced Po and I' by increasing channel transitions to the closed state and reducing To without affecting Imax. The presence of diazoxide, minoxidil or cromakalim on the cytoplasmic side of the channel did not prevent [ATP]cis or [glibenclamide]cis from blocking the channel. The data suggest that the action(s) of these drugs are not due to their effects on the phosphorylation of the channel protein. The glibenclamide- and cromakalim-induced effects on the SCl channel are mediated via a "flicker" type block mechanism. Modulation of the SCl channel by [diazoxide]cis and [glibenclamide]cis highlights the therapeutic potential of these drugs in regulating the Ca2+-counter current through this channel.     

Lack of response of the rat liver "class 3" cytosolic aldehyde dehydrogenase to toxic chemicals, glutathione depletion, and other forms of stress

One of the rat liver "Class 3" cytosolic aldehyde dehydrogenases (EC 1.2.1.3), ALDH3c, is known to be markedly induced by polycyclic aromatic hydrocarbons and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD; dioxin). In the present study we examined whether hepatic ALDH3c induction is a general response to toxicity. Treatment of Wistar rats for 4 days with known toxic doses of hepatotoxic agents--carbon tetrachloride, dimethylnitrosamine, diethylnitrosamine, aflatoxin B1, and D-ethionine--did not induce ALDH3c enzyme activity. Whereas dimethylaminoazobenzene at 100 mg/kg/day for 4 days did not increase ALDH3c, a 10-fold lower dose of dimethylaminoazobenzene for 4 days produced a 20-fold increase in ALDH3c activity. Treatment with phorone, diethylmaleate or L-buthionine-S,R-sulfoximine--which deplete reduced glutathione (GSH) by different mechanisms--did not affect ALDH3c activity. One dose of benzo[a]pyrene for 24 hr increased ALDH3c activity by 25-fold. Treatment with both the GSH-depleting chemicals and benzo[a]pyrene inhibited ALDH3c induction by 45% to 75%, suggesting a role for GSH during ALDH3c induction. After ALDH3c activity had already been induced by benzo[a]pyrene, however, the GSH-depleting chemicals did not affect ALDH3c activity. No changes in ALDH3c activity were seen 24 or 48 hr after partial hepatectomy, on the fifth day following surgical cholestasis, or after guanethidine-induced sympathectomy. These data indicate that hepatic ALDH3c inducibility in the rat is not a general or direct response to chemical toxicity, or to conditions of GSH depletion or other forms of stress.     

Effects of sodium bicarbonate administration on the exercise tolerance of normal subjects breathing through dead space

Granulocyte-macrophage colony-stimulating factor (GM-CSF)-transduced B16-F10 murine melanoma cells had lower tumorigenicity in both syngeneic and nude mice than parental or LacZ-transduced (control) cells. The subcutaneous (s.c.) tumors producing GM-CSF were densely infiltrated with macrophages, whereas the control tumors were not. In vitro studies showed that GM-CSF-transduced B16 cells were susceptible to lysis mediated by nonactivated murine macrophages, whereas control B16 cells were not. Macrophage-mediated cytotoxicity against GM-CSF-transduced B16 cells was independent of the presence of NO, H2O2, O2-, tumor necrosis factor alpha, and matrix metalloproteinase. Coculture experiments using GM-CSF-producing and -nonproducing B16 cells demonstrated that GM-CSF produced by the transduced B16 cells activated macrophages to kill the bystander non-GM-CSF-producing tumor cells. The results suggest that GM-CSF released by tumor cells can induce macrophage-mediated cytotoxicity, which in turn can inhibit the in vivo growth of GM-CSF-transduced tumor cells.     

Modifications of p53 protein and accumulation of p21 and gadd45 mRNA in TGF-beta 1 growth inhibited cells

Transforming growth factory beta (TGF-beta) is a potent growth inhibitor of epithelial cells. One of the strategies used to elucidate the anti-proliferative mode of action of TGF-beta is to find out whether the receptor-generated signals interact with components of the basic machinery of the cell cycle. In this study we examined whether p53 and two other cycle inhibitory genes that can be transactivated by p53 are affected by TGF-beta 1 in epithelial cells. We show that TGF-beta 1 signalling controls the intracellular localization as well as the phosphorylation pattern and the stability of p53 protein. TGF-beta signalling also elevates the expression of p21/waf-1 and gadd45. The observed modifications in the protein suggest that p53 is involved in mediation of TGF-beta 1 growth inhibition. However, in TGF-beta 1 growth inhibited cells, wild type p53 is not required for the accumulation of the two p53 downstream targets p21/waf-1 and gadd45.     

Tissue response to covered Wallstents

8-epi prostaglandin F2alpha(8-epi PGF2alpha) contracted rat thoracic aorta rings in a concentration-dependent manner in the presence or absence of functional endothelium [median effective concentration (EC50) values, 455+/-52 and 268+/-34 nM, respectively; Student's t test; p=0.006]. U46619 was a more potent agonist with or without functional endothelium (EC50 values, 6.8+/-1.6 and 4.5+/-1.0 nM, respectively). SQ29548 [a thromboxane (TP)-receptor antagonist] inhibited contractions to both 8-epi PGF2alpha and U46619 in a competitive manner, with mean pA2 values of 8.3 and 7.9, respectively. 8-Epi PGF2alpha had a further contractile effect in vessels that had been contracted with noradrenaline and had been shown to possess a functional endothelium. Inhibition of thromboxane synthesis with OKY-046 or blockade of endothelin receptors with bosentan had no effect on responses to 8-epi PGF2alpha or U46619. Preincubation with 8-epi PGF2alpha or noradrenaline shifted the concentration-response curves to U46619 upward at low concentrations of U46619 with no significant change in EC50 values or maximal responses. Reduction of TP-receptor number in rat aorta with dithiothreitol caused a concentration-dependent inhibition of responses to both U46619 and 8-epi PGF2alpha, with no effect on maximal responses and or on the responses to U46619 after the preincubation with 8-epi PGF2alpha. These results indicate that 8-epi PGF2alpha is a potent vasoconstrictor in the rat aorta and are suggestive of an action of 8-epi PGF2alpha at the TP receptor.     

Tumor cell heterogeneity: impact on mechanisms of therapeutic drug resistance

PURPOSE: The aim of these studies was to determine whether chemotherapy-resistant tumor cell sublines derived from a single starting cell population with identical treatment protocols, have the same mechanism of resistance. METHODS AND MATERIALS: Twelve cyclophosphamide-resistant sublines were derived from KHT-iv murine sarcoma cells by repeated exposures to 2, 4, or 8 microg/ml doses of 4-hydroperoxycyclophosphamide (4-OOHCP). To investigate possible mechanisms of resistance, glutathione (GSH) levels, glutathione S-transferase (GST) activity, and aldehyde dehydrogenase (ALDH) activity were determined. In addition, studies with the GSH depletor buthionine sulfoximine (BSO) and the ALDH inhibitor diethylamino-benzaldehyde (DEAB) were undertaken. RESULTS: Resistant factors to 4-OOHCP, assessed at 10% clonogenic cell survival, ranged from 1.5-7.0 for the various cell lines. Crossresistance to melphalan and adriamycin also were commonly observed. Increased GSH levels, GST activity and ALDH activity were detected in the sublines but not all exhibited the same pattern of biochemical alterations. The response to GSH and ALDH inhibitors also varied among the sublines; the resistance being reversible in some cell lines but not others. CONCLUSION: The present results indicate that when resistant sublines are derived simultaneously from the same starting cell population, the observed mechanisms of resistance may not be the same in each of the variants. These findings support the hypothesis that preexisting cellular heterogeneity may affect mechanisms of acquired resistance.     

Effects of electrode configuration on psychophysical strength-duration functions for single biphasic electrical stimuli in cats

The interface between electrode and neural target tissue is thought to influence certain characteristics of neural and behavioral responses to electrical stimulation of the auditory system. At present, the biophysical properties of this interface are not well understood. Here the effects of biphasic phase duration and electrode configuration on psychophysical threshold in response to electrical stimulation in cats are described. Five cats were trained to respond to acoustic stimuli using food as a reward in an operant reinforcement paradigm. After training, the animals were unilaterally deafened and implanted with a multicontact intracochlear electrode array. Thresholds for single presentations of biphasic current pulses were measured as a function of phase duration and electrode arrangement. Statistical analyses of the data indicated that strength-duration function slopes between 200 and 1600 microseconds/phase were significantly different for the different electrode configurations and, overall, were unrelated to the absolute level of the strength-duration function (i.e., were independent of absolute threshold). For all subjects, the slope of this function for intermediate pulse durations was dependent on electrode configuration and most shallow for radial-bipolar configurations (-3.4 dB/doubling), was steepest for monopolar arrangements (-5.9 dB/doubling), and was intermediate for longitudinal-bipolar pairings. (-4.4 dB/doubling). Slopes for both shorter and longer phase duration stimuli were not significantly different. The underlying mechanisms for these effects may include, or be a combination of altered electrical field patterns, integrated activity across multiple fibers, and stochastic behavior of individual auditory neurons to electrical stimulation.