Ethanol-Induced Enhancement of Cocaine Bioactivation and Irreversible Protein Binding: Evidence Against a Role of Cytochrome P-450IIE1

Chronic ethanol consumption potentiates cocaine-induced liver injury in rodents. Since cocaine has to be bioactivated by a cytochrome P-450-dependent N-oxidative pathway to exert its hepatotoxic effects, we studied the role of the ethanol-inducible P-450IIE1 for cocaine metabolism. Male Sprague-Dawley rats were pretreated with either a liquid diet containing ethanol (30% of calories) for 4 weeks or injected with pyrazole (200 mg/kg/day, ip, for 3 days). Both agents induced microsomal p-nitrophenol hydroxylation which is a probe for the catalytic activity of P-450IIE1. However, only ethanol, but not pyrazole, increased both microsomal cocaine N-demethylase activity (by 47%) and the extent of irreversible binding of [3H]-cocaine to microsomal proteins (by 100%), which was taken as a quantitative endpoint for the formation of a reactive metabolite. Cocaine N-demethylation and irreversible protein binding of cocaine were not inhibited by P-450IIE1 isozyme-selective substrates, nor was the rate of cocaine metabolism and binding decreased by functionally active polyclonal anti-rat P-450IIE1 antibodies. Furthermore, pyrazole pretreatment sensitized cultured hepatocytes to the glutathione-dependent cytotoxic effects of nontoxic concentrations of cocaine. These results indicate that (a) cocaine is not a major substrate for the ethanol-inducible P-450IIE1, (b) the enhancing effects of ethanol on cocaine bioactivation may be due to induction of other P-450 isoforms, and (c) induction of P-450IIE1 may potentiate cocaine-induced hepatocellular toxicity in vitro independently of cocaine metabolism, e.g., by P-450IIE1-dependent oxidative stress.     

Expression and regulation of connexins in cultured ventricular myocytes isolated from adult rat hearts

Gap junctions were assayed during re-differentiation of adult rat cardiomyocytes in long-term culture to gain insight into the processes of remodeling. Double immunostaining allowed the localization of connexins Cx40, Cx43, and Cx45 between myocytes and demonstrated co-expression and co-localization in individual cells and gap junction plaques, respectively. Immunoblots showed differential time-dependent changes in connexin expression and phosphorylation. The total amount of connexins and the ratio of phosphorylated/non-phosphorylated isoforms gradually increased during the re-establishment of intercellular communication. Dual voltage-clamp studies showed the involvement of several types of gap junction channels. Multichannel currents yielded diverse spectra of g(j,inst)=f( V(j)) and g(j,ss)=f( V(j)) relationships ( g(j,inst): instantaneous gap junction conductance; g(j,ss): conductance at steady state; V(j): transjunctional voltage), indicative of homotypic and heterotypic channels. Single-channel currents revealed two prominent conductances reflecting gamma(j,main) and gamma(j,residual). The histograms of gamma(j,main) showed four discrete peaks (41-44, 59-61, 70-76, and 100-107 pS) attributable to a combination of Cx45-Cx45, Cx40-Cx45 and Cx43-Cx45 channels (1st peak), Cx43-Cx43 and Cx40-Cx43 channels (2nd peak), Cx43-Cx43 channels (3rd peak) and Cx40-Cx40 and Cx40-Cx43 channels (4th peak). However, the presence of heteromeric channels cannot be excluded. The data are consistent with an up-regulation of Cx45 and Cx43 during re-differentiation.     

IP-10 and type 1 diabetes: a question of time and location

Chemokines are key signal molecules that attract cells of the host immune system to the site of a potential threat. Virus infections usually induce a massive chemokine and cytokine burst and therefore recruit a large plethora of leukocytes to the site of infection with the goal to restrict and abrogate viral spread. The down side of this massive excitation of the human defense system is non-specific activation of potentially self-reactive lymphocytes. Coupled with an antigen-specific event, for example molecular mimicry between host components and viral proteins, autoimmunity might be the consequence in susceptible individuals. However, activated immune components with autoaggressive potential must find their target and must remain in one site sufficiently long in order to cause chronic tissue damage. In this review we will focus on the influence of the chemokine IP-10 (CXCL10) on the trafficking of autoaggressive cells during the immunopathogenesis of type 1 diabetes (T1D) and explain why IP-10 can have a dual effect on T1D depending on time and location of expression.     

Efficiency of work performance and contraction velocity in isotonic tetani of frog sartorius

Mechanical work, ATP, ADP, PC, free creatine and lactate concentrations were determined on IAA poisoned frog sartorii tetanically stimulated in humidified N, at 10°C in isotonic conditions (0.25 or 0.45 P₀). Tetanus duration was 0.35 s, number of tetani was varied from 0 (rest) to 25 (exhaustion). The mechanical work performed per mole ATP+PC split (W _P ^* ) amounted on the average to 16.7 kJ/mol. It was observed, however, that w _p ^* increased from about 13 to about 24 kJ/mol with decreasing ATP concentration from about 2 (resting value) to about 1 mol/g and that this decrease in ATP was associated with a decrease of the shortening (and relaxation) speed of the muscle to about 30% of the values observed on the first tetanus. It is concluded that the thermodynamic efficiency of muscle contraction, calculated from the ratio of w _P ^* (measured) to the thermodynamic affinity (free energy change) of ATP hydrolysis (estimated) increases from about 0.3 to about 0.5 with decreasing ATP concentration and shortening speed.This work was supported by the Fonds National Suisse de la Recherche Scientifique, Grants 3.332.78 and 3.364.0.82     

Disruption of mCry2 restores circadian rhythmicity in mPer2 mutant mice

Many biochemical, physiological, and behavioral processes display daily rhythms generated by an internal timekeeping mechanism referred to as the circadian clock. The core oscillator driving this clock is located in the ventral part of the hypothalamus, the so called suprachiasmatic nuclei (SCN). At the molecular level, this oscillator is thought to be composed of interlocking autoregulatory feedback loops involving a set of clock genes. Among the components driving the mammalian circadian clock are the Period 1 and 2 (mPer1 and mPer2) and Cryptochrome 1 and 2 (mCry1 and mCry2) genes. A mutation in the mPer2 gene leads to a gradual loss of circadian rhythmicity in mice kept in constant darkness (DD). Here we show that inactivation of the mCry2 gene in mPer2 mutant mice restores circadian rhythmicity and normal clock gene expression patterns. Thus, mCry2 can act as a nonallelic suppressor of mPer2, which points to direct or indirect interactions of PER2 and CRY2 proteins. In marked contrast, inactivation of mCry1 in mPer2 mutant mice does not restore circadian rhythmicity but instead results in complete behavioral arrhythmicity in DD, indicating different effects of mCry1 and mCry2 in the clock mechanism     

Fiber optic probe for polarized reflectance spectroscopy in vivo: design and performance

We present the design and construction of a fiber optic probe for elastic light scattering spectroscopy in vivo with polarized excitation and polarization sensitive detection. The performance of the fiber probe is evaluated using a suspension of polystyrene spheres placed atop a diffusely scattering substrate, and it demonstrates that the size-dependent characteristics of the scatterers can be extracted in the presence of a highly diffusely scattering background using a linear combination of forward and backward Mie scattering components of the scatterers. Subsequently, Mie theory calculations are performed over a broad range of diagnostically relevant parameters of nuclei-mean diameter, size distribution, and relative refractive index-to understand how the polarized reflectance measurements with the fiber probe can be used to extract morphological information about epithelial tissue. Finally, the feasibility of in vivo measurements with the fiber optic based polarization sensitive light scattering spectroscopy is demonstrated.     

Fiber optic probes for biomedical optical spectroscopy

Fiber optic probes are a key element for biomedical spectroscopic sensing. We review the use of fiber optic probes for optical spectroscopy, focusing on applications in turbid media, such as tissue. The design of probes for reflectance, polarized reflectance, fluorescence, and Raman spectroscopy is illustrated. We cover universal design principles as well as technologies for beam deflecting and reshaping.     

Sex differences in emotional and psychophysiological responses to musical stimuli.

Although it is known that men and women differ in their music preferences and emotional reactions to music, little is known about sex differences in physiological reactions to music. In our study, we therefore set out to examine the differential reactivity to two musical stimuli that elicit distinct psychological and physiological reaction patterns. Fifty-three healthy subjects (mean age: 26.13, SD: 3.97; 26 males, 27 females) were examined. Heart rate, electrodermal activity, skin temperature, salivary cortisol, salivary alpha-amylase, and psychological variables were assessed during the course of the whole study. Following baseline assessment, two musical stimuli, which were carefully selected and rated in a pre-study as relaxing and pleasant (renaissance music) and arousing and unpleasant (heavy metal), respectively, were introduced. They were presented on two different days in a randomized order. Whereas psychological variables did not differ between men and women, results of electrophysiological measures indicate significantly different reactivity patterns between men and women. Women displayed elevated response curves to the arousing and unpleasant stimulus, whereas men did not. However, no differences were found with regards to endocrine measures in saliva. Our results demonstrate sex differences in reactivity patterns to musical stimuli in psychophysiological measures. In our study, we were able to show that women tend to show hypersensitivity to aversive musical stimuli. This finding is in accordance with previous literature on sex differences in emotion research. Furthermore, our study indicates that the confounding effects of sex differences have to be considered when using musical stimuli for emotion induction.