bluuebunny/crossref_metadata_2025 huggingface dataset:
Transcriptional Activity of the Tandem Repeat Polymorphism in the 5′‐Flanking Region of the Human CYP2E1 Gene
Authors: Nomura Fumio , Itoga Sakae , Uchimoto Takayuki , Tomonaga Takeshi , Nezu Masahiko , Shimada Hideaki , Ochiai Takenori
Published: November 2003

Background: There are remarkable interindividual differences in the expression of cytochrome P‐4502E1(CYP2E1), which in turn may alter susceptibility to alcohol‐related diseases and various cancers. We recently characterized the tandem repeat polymorphism in the 5′‐flanking region of the human CYP2E1 gene and found that subjects with the homozygous mutant‐type (A4/A4) may be at higher risk of developing esophageal cancer. In this study, we determined how this tandem repeat polymorphism alters transcriptional activities of the human CYP2E1 gene by transfection studies.

Methods: The 5′‐flanking region (−2562 base pair to +9 base pair) of the CYP2E1 gene from three individuals of different genotypes (A2/A2, A2/A4, A4/A4) was amplified by polymerase chain reaction. The polymerase chain reaction products placed in front of a luciferase reporter gene were transfected into human hepatoblastoma cells, human esophageal cancer cells, and human uterus cancer cells. Transcriptional activities were determined by the dual‐luciferase assay. When indicated, ethanol (50 mM) was included in the culture medium. CYP2E1 messenger RNA levels in peripheral lymphocytes were measured by the real‐time reverse transcription‐polymerase chain reaction using the LightCycler system.

Results: The construct including the tandem repeat region exhibited luciferase activities in both A2 and A4 type. It was of note that the activity produced by the A4 allele was significantly greater than that by A2 allele in HeLa cells (p < 0.001). CYP2E1 messenger RNA levels in peripheral blood lymphocytes were comparable between the two genotypes.

Conclusion: Transcriptional activity of the mutant allele of the tandem repeat polymorphism in the 5′‐flanking region of the CYP2E1 gene is greater than that of the wild type.
Augmentation of Ethanol‐Induced Cell Damage and Activation of Nuclear Factor‐κB by Annexin IV in Cultured Cells
Authors: Sohma Hitoshi , Ohkawa Hiroko , Sakai Rie , Hashimoto Eri , Ukai Wataru , Saito Toshikazu
Published: November 2003

Background: We previously reported that the amount of annexin IV expression was increased in culture cells by exposure to ethanol. Here, to investigate the physiologic role of annexin IV, we analyzed ethanol‐induced cytotoxicity and nuclear factor (NF)‐κB activity by using annexin IV‐overexpressed cells.

Methods: Annexin IV overexpression was performed by transfection of expression vector, in which annexin IV complementary DNA was ligated, to culture cells (rat glioma C6 cells and human neuroblastoma SH‐SY5Y cells). Ethanol‐induced cytotoxicity was assayed by measuring the mitochondrial enzyme (dehydrogenase) activity or trypan blue exclusion. NF‐κB activity was measured by electrophoretic mobility shift assay with a κB‐oligonucleotide probe.

Results: Ethanol‐induced cytotoxicity was increased by overexpression of annexin IV in both C6 cells and SH‐SY5Y cells. Annexin IV overexpression augmented ethanol‐induced NF‐κB activation.

Conclusions: Ethanol‐induced increase in annexin IV expression might amplify ethanol‐induced cytotoxicity via NF‐κB activation.
Ethnicity and Psychiatric Comorbidity Among Alcohol‐Dependent Persons Who Receive Inpatient Treatment: African Americans, Alaska Natives, Caucasians, and Hispanics
Authors: Hesselbrock Michie N. , Hesselbrock Victor M. , Segal Bernard , Schuckit Marc A. , Bucholz Kathy
Published: November 2003

This study examined ethnic and gender differences of psychiatric comorbidity among alcohol dependent men and women from four ethnic groups: Alaska Native, Caucasians, African Americans, and Hispanics. The data were obtained through individual standardized interview; DSM‐III‐R diagnoses were obtained via a computer algorithm. The subjects inclued 1177 Caucasians, 361 African Americans, 93 Hispanics and 486 Alaska Natives. Significant ethnic differences were found in relation to age of onset of alcohol and multiple substance dependence and psychiatric comorbidity. Ethnic differences were also noted with regard to the health care utilizations.
Interaction Between S‐Propranolol and Ethanol in Mice Selectively Bred for Ethanol Sensitivity: The Inbred Short‐ and Long‐Sleep Mice
Authors: Deitrich Richard A. , Bludeau Pequita
Published: November 2003

Background: We have studied the effect of a β‐adrenergic blocking agent, S‐propranolol, on the response of mice to anesthetic doses of ethanol. We used the selectively bred short and long sleep (ISS and ILS) mice. These mice were selected for their differential sensitivity to anesthetic doses of ethanol and then inbred. The study was prompted by the finding that the effect of ethanol on the firing rate of cerebellar Purkinje cells is modulated by β‐adrenergic input. In addition, this firing rate depression by ethanol is highly correlated with the anesthetic potency of ethanol. We were attempting to find a behavioral correlate of this effect of β‐adrenergic agents in the ISS and ILS mice.

Methods: We studied the effect of S‐propranolol plus ethanol on the sleep time and blood ethanol at awakening in the inbred ILS and ISS mice. We administered anesthetic doses of ethanol with and without S‐propranolol. We conducted studies of the rate of disappearance of ethanol in the presence of S‐propranolol and carried out sleep time and metabolic studies with mice in an incubator held at 32 to 33°C.

Results: We found that S‐propranolol caused a prolonged anesthetic time brought about by ethanol but only in ISS mice. There was no significant difference in the blood ethanol levels at awakening with or without S‐propranolol, indicating that S‐propranolol had no effect on the brain sensitivity. Subsequently, we showed that this was due to a profound hypothermia caused by a combination of S‐propranolol and ethanol. This was greater in the ISS mice because a larger dose of ethanol was required for the anesthetic effect of ethanol. The effect on ethanol disappearance rate, temperature drop, and anesthesia time all were largely reversed by placing the animals in an incubator at 32 to 33°C.

Conclusions: Profound hypothermia lowers the ethanol disappearance rate when both S‐propranolol and ethanol are given. The effect of S‐propranolol is likely due to the blockade of β‐adrenergic receptors that prevents thermogenic responses to the hypothermia brought about by ethanol. The results indicated that there might be a genetic effect controlling the hypothermic response to the combination of S‐propranolol and ethanol. Further experiments to investigate this are reported in a subsequent article. We could find no evidence of a central nervous system effect of S‐propranolol on the hypnotic actions of ethanol in these strains of mice.
Ethnic Differences in Driving After Drinking and Riding With Drinking Drivers Among Adolescents
Authors: Walker Samantha , Treno Andrew J. , Grube Joel W. , Light John M.
Published: November 2003

Background: This study examined the relationship between ethnicity and driving after drinking (DD) and riding with drinking drivers (RWDD) while controlling for drinking patterns, driving practices, and background demographic characteristics including age and gender.

Methods: Using random‐digit dialing procedures, 1534 young adults ranging from 15‐ to 20‐years of age(mean = 17.6) living in California were recruited to participate in a telephone survey. Latinos, African Americans, and Asian Americans were oversampled to allow cross‐group comparisons.

Results: Rates of DD were lower for females than for males and were also lower for African Americans and Asian Americans than for whites. However, after we controlled for drinking patterns and driving practices, the results showed Latinos at greater risk for DD than white adolescents. Compared with whites and males, Asian American and female adolescents were less likely to report RWDD. When drinking patterns and driving practices were taken into account, Latino adolescents were nearly twice as more likely to ride with drinking drivers than whites.

Conclusions: These findings indicate a greater need for directing prevention efforts to target Latino youth and youth at risk. Moreover, research aimed at elucidating the social and environmental factors involved in the low prevalence rates of DD and RWDD among Asian American youth may indicate possible protective factors to DD and RWDD operating within the Asian American community.
Short‐Term Selection for the Interaction Between S‐Propranolol and Ethanol in Mice
Authors: Deitrich Richard A. , Bludeau Pequita
Published: November 2003

Background: We have previously demonstrated that there is an interaction between S‐propranolol, a β‐adrenergic blocking agent, and ethanol on the hypnotic sensitivity of inbred short‐ and long‐sleep mice (ISS and ILS). We found that the interaction was due to an additive hypothermic effect of ethanol and S‐propranolol that markedly decreased the disappearance rate of ethanol. There was no discernible effect of S‐propranolol on the hypnotic actions of ethanol as evidenced by the waking blood ethanol levels. Because ISS mice were more sensitive to this effect than were ILS mice, it seemed that there was a genetic effect to the response. Therefore, we carried out a short‐term breeding project to determine whether the response could be selectively bred.

Methods: We used the heterogeneous stock of mice from the Institute for Behavioral Genetics to carry out the selection study. The index for selection was the difference in sleep time between the same animal treated with propranolol and ethanol versus treatment with saline and ethanol at least 1 week apart.

Results: In four generations, we were able to achieve separation between mice with a large difference in sleep time (high line) from those with a smaller difference in sleep (low line) time. There was no difference between the average blood ethanol at awakening in the high line versus the low line. The effect was not due to a difference in rate of propranolol metabolism between the two lines. Sleep time with ethanol alone was not different between the high and low lines.

Conclusions: The magnitude of the interaction between ethanol and S‐propranolol is controlled by alleles with polymorphisms present in the HS stock of mice. The response is most likely due to a difference in the mechanisms of thermogenesis controlled by the β‐adrenergic receptors in muscle and fat. Because there were no sleep time differences between the high and low lines given ethanol alone, central nervous system sensitivity to ethanol is not a correlated response to the selection.
The Cyclic AMP/Protein Kinase A Signal Transduction Pathway Modulates Tolerance to Sedative and Hypothermic Effects of Ethanol
Authors: Yang Xioaju , Oswald Lynn , Wand Gary
Published: November 2003

Background: An expanding body of literature indicates the important role of the cAMP/PKA signaling pathway in establishing initial sensitivity to alcohol as well as being involved in certain forms of tolerance to ethanol. The use of mice with heterozygous inactivation of the Gnas gene encoding Gsα allowed us to explore the relationship between tolerance to ethanol and cAMP/PKA signaling.

Methods: Mice with the targeted disruption of one Gsα allele were compared with wild‐type littermates in their initial sensitivity to ethanol‐induced sedation and hypothermia and then monitored for the development of tolerance during two subsequent bouts of intoxication. Components of the cAMP/PKA signaling pathway were analyzed in ethanol‐naïve mice and again following the development of tolerance to ethanol to better understand the contribution of this signaling pathway to the acquisition of tolerance.

Results: During the initial exposure to ethanol, mice with the targeted disruption of one Gsα allele (Gnas) were more sensitive to the sedative effects of ethanol compared with wild‐type littermates. Wild‐type mice developed within‐session tolerance to ethanol‐induced hypothermia whereas Gnas mice did not. Following the subsequent ethanol treatments, wild‐type mice developed between‐session tolerance to the sedative effects of ethanol to a greater degree than mice with heterozygous inactivation of the Gnas gene. The development of tolerance to the sedative effects of ethanol was accompanied by increased expression of phospho‐CREB in the cerebellum, hippocampus, and frontal cortex. No changes in phospho‐CREB expression were detected in these brain regions in mice with heterozygous inactivation of the Gnas gene.

Conclusion: The results show that cAMP/PKA signal transduction modulates sensitivity to sedative and hypothermic effects of ethanol. This signal transduction system also influences the acquisition of within‐session and between‐session tolerance. The mechanism through which cAMP/PKA signaling modulates the development of tolerance remains to be elucidated but may involve changes in phospho‐CREB expression.
Tobacco Use is Associated With Reduced Central Serotonergic Neurotransmission in Type 1 Alcohol‐Dependent Individuals
Authors: Berggren Ulf , Fahlke Claudia , Eriksson Matts , Balldin Jan
Published: November 2003

Background: Reduced central serotonergic neurotransmission in alcohol dependence may be attributed to the effects of cigarette smoking (and possibly more specifically to nicotine) rather than to alcoholism or its subtypes. The aim of the present study was therefore to compare central serotonergic neurotransmission in tobacco‐using (cigarette smokers and users of smokeless tobacco, i.e., snuffers) alcohol‐dependent individuals to that of tobacco‐nonusing alcohol‐dependent individuals.

Methods: The central serotonergic neurotransmission was assessed by the prolactin (PRL) response to the serotonin‐releasing agent D‐fenfluramine (30 mg orally). Male subjects (n= 37) aged 20–65 years were recruited for this purpose. They were all type 1 alcohol‐dependent individuals and had ended their alcohol intake the day before the D‐fenfluramine challenge test.

Results: There was no difference in baseline PRL concentrations between tobacco‐using (n= 18) and tobacco‐nonusing (n= 19) alcohol‐dependent individuals. On the other hand, the maximum PRL response after D‐fenfluramine was significantly lower in the tobacco‐using group as compared to the tobacco‐nonusing individuals.

Conclusion: Whether the reduction in central serotonergic neurotransmission in tobacco‐using alcohol‐dependent individuals is pre‐existing or a result of tobacco use remains to be elucidated.
Expression of hNP22 Is Altered in the Frontal Cortex and Hippocampus of the Alcoholic Human Brain
Authors: Depaz Iris , Ito Masayuki , Matsumoto Izuru , Niwa Shin‐ichi , Kroon Paul , Wilce Peter A.
Published: November 2003

Background: Human neuronal protein (hNP22) is a gene with elevated messenger RNA expression in the prefrontal cortex of the human alcoholic brain. hNP22 has high homology with a rat protein (rNP22). These proteins also share homology with a number of cytoskeleton‐interacting proteins.

Methods: A rabbit polyclonal antibody to an 18‐amino acid epitope was produced for use in Western and immunohistochemical analysis. Samples from the human frontal and motor cortices were used for Western blots (n= 10), whereas a different group of frontal cortex and hippocampal samples were obtained for immunohistochemistry (n= 12).

Results: The hNP22 antibody detected a single protein in both rat and human brain. Western blots revealed a significant increase in hNP22 protein levels in the frontal cortex but not the motor cortex of alcoholic cases. Immunohistochemical studies confirmed the increased hNP22 protein expression in all cortical layers. This is consistent with results previously obtained using Northern analysis. Immunohistochemical analysis also revealed a significant increase of hNP22 immunoreactivity in the CA3 and CA4 but not other regions of the hippocampus.

Conclusions: It is possible that this protein may play a role in the morphological or plastic changes observed after chronic alcohol exposure and withdrawal, either as a cytoskeleton‐interacting protein or as a signaling molecule.
Longitudinal Model Predicting Partner Violence Among White, Black, and Hispanic Couples in the United States
Authors: Field Craig A. , Caetano Raul
Published: November 2003

Background: A limited amount of information pertaining to ethnic‐specific risk factors associated with intimate partner violence across time currently exists. The current study examines ethnic‐specific longitudinal predictors of male‐to‐female and female‐to‐male partner violence (MFPV and FMPV) in white, black, and Hispanic couples in the U.S. general population.

Methods: In 1995, a total of 1635 married or cohabitating couples 18 years of age or older living in households in the 48 contiguous states participated in a national survey that used a multistage probability sampling procedure with 100 primary sampling units and included oversamples of blacks and Hispanics. In 2000, the follow‐up survey had an overall response rate of 72% and included 406 white, 232 black, and 387 Hispanic intact couples.

Ethnic‐specific regression models predicting MFPV and FMPV at follow‐up were developed. The risk factors of interest included male and female reports of history of childhood abuse, exposure to parental violence, impulsivity, alcohol problems, frequency of drinking five or more drinks per occasion, volume of alcohol consumed per week in average standard drinks, approval of marital aggression and male‐to‐female and female‐to‐male partner violence at baseline. MFPV and FMPV in 1995 and 2000 were based on the Conflict Tactics Scale Form R.

Results: Black and Hispanic couples were at approximately three times greater risk of MFPV and two times greater risk of FMPV at follow‐up in comparison to white couples even after controlling for sociodemographic characteristics, alcohol consumption, and psychosocial variables. Extreme specific models indicated that among blacks, MFPV was a significant predictor of MFPV and FMPV at follow‐up. In contrast, among Hispanics, FMPV was a significant predictor of FMPV and MFPV at follow‐up.

Conclusions: Ethnic‐specific multivariate logistic regression models indicated that the predictors of MFPV and FMPV including psychosocial variables, alcohol use, and alcohol‐related problems varied by ethnicity. These findings contribute to our continually growing knowledge base regarding ethnic differences associated with the development of intimate partner violence and have important implications for prevention and intervention.
Dopamine Activity in the Nucleus Accumbens During Consummatory Phases of Oral Ethanol Self‐Administration
Authors: Doyon William M. , York Jennifer L. , Diaz Laurea M. , Samson Herman H. , Czachowski Cristine L. , Gonzales Rueben A.
Published: November 2003

Background: This present study was designed to clarify the role of dopamine in the nucleus accumbens during operant ethanol self‐administration by separating bar pressing (ethanol seeking) from ethanol consumption. Furthermore, we sought to define the relationship between ethanol in the brain and the accumbal dopamine response after oral self‐administration of ethanol.

Methods: Two separate groups of male Long‐Evans rats were trained to bar press with 10% ethanol or water. Rats were trained to elicit an escalating number of bar presses across daily sessions before gaining access to the drinking solution for 20 min. Microdialysis was performed before (during a waiting period), during, and after bar pressing and drinking. A handling control group was included, but did not receive training.

Results: A significant increase in dopamine occurred during placement of the rats into the operant chamber in trained rats and handling controls. The lever‐pressing period did not produce an increase in dialysate dopamine. Accumbal dopamine was increased in the first 5 min of ethanol, but not water, consumption. Ethanol appeared in the dialysate sample following ethanol availability, and peak concentrations were reached at 10 min. Most of the ethanol and water consumption occurred within 5 min of fluid access. The probes were distributed in the core (32%), shell (32%), and core plus shell (36%) regions of the nucleus accumbens.

Conclusions: The enhancement of dopamine during transfer into the operant chamber does not depend on anticipation or operant training with ethanol or water reinforcement. Furthermore, the difference between the time course of accumbal dopamine and ethanol in dialysates suggests that the dopamine response is not solely due to pharmacological effects of ethanol. The dopamine response may be associated with the stimulus properties of ethanol presentation, which would be strongest during consumption.
Reinforcement From Pharmacological Effects of Ethanol in Newborn Rats
Authors: Petrov Evgeniy S. , Varlinskaya Elena I. , Spear Norman E.
Published: November 2003

Background: The preweanling period in the rat is characterized by acceptance of substantial amounts of ethanol and susceptibility to its reinforcing effects. It has been unclear, however, whether the neurobiological basis of ethanol reinforcement properties at this age is in ethanol's olfactory, gustatory, or pharmacological effects.

Methods: The effectiveness of intraperitoneal (ip) ethanol as a reinforcer for newborn (3‐hr‐old) rats was tested toward separation of the orosensory and pharmacological sources of ethanol reinforcement. Responsiveness to a test nipple by pups given such pairings was compared with that of pups given unpaired presentations of the nipple and ethanol.

Results: Reinforcement was assessed in terms of response to a surrogate nipple 1 hr after a single pairing of a similar nipple providing water (conditioned stimulus) and ip injection of ethanol (0.125, 0.25, 0.50, or 0.75 g/kg; unconditioned stimulus). Significant effects of ethanol reinforcement occurred with the lower doses (0.125 and 0.25 g/kg); higher doses of ethanol (0.50 and 0.75 g/kg) had no significant reinforcement effect. A second experiment determined that for conditioning with ip ethanol as the unconditioned stimulus, a conditioned stimulus consisting of only ingesting water or only suckling on an empty nipple also yielded significant reinforcing effects of ethanol, although with less strength than their combination. Both reinforcing doses of ethanol, 0.125 and 0.25 g/kg, yielded detectable concentrations of ethanol in the blood 5 min after injection, which were sustained at a significantly lower level 60 min after administration.

Conclusions: These data indicate that aside from possible, and likely weak, hematogenic sources of gustatory and olfactory attributes of ethanol, the basis of ethanol's reinforcement effect in neonatal rats is primarily pharmacological. For the pharmacological effects of ethanol to be reinforcing for the neonatal rat, concurrent appetitive activity on a nipple providing a fluid may be necessary for a substantial effect with this paradigm.
Chronic Intermittent Injections of High‐Dose Ethanol During Adolescence Produce Metabolic, Hypnotic, and Cognitive Tolerance in Rats
Authors: Silvers Janelle M. , Tokunaga Sayaka , Mittleman Guy , Matthews Douglas B.
Published: November 2003

Background: Many humans are first exposed to ethanol during adolescence, the time at which they are most likely to binge drink ethanol. Chronic intermittent ethanol (CIE) exposure produces ethanol tolerance in adolescent rodents. Recent studies suggested that adolescent animals administered CIE experienced increased cognitive impairment following an ethanol challenge. These studies further explore development of ethanol tolerance caused by CIE in adolescence, and whether CIE during adolescence leads to altered ethanol response in adulthood.

Methods: Beginning postnatal day (P) 30, adolescent rats were administered 5.0 g/kg ethanol or saline every 48 hours for 20 days. In experiment I, animals were tested for differential weight gain. In experiment II, loss of righting reflex (LORR) was observed after each injection, then at completion of pretreatment all animals were tested with 5.0 g/kg ethanol and LORR was observed. In experiment III, blood ethanol levels were observed and elimination rates calculated after the first and fifth pretreatments. All animals were tested with 5.0 g/kg at completion of pretreatment and elimination rates were recalculated. In experiment IV, animals were trained on the spatial version of the Morris Water Maze Task (MWMT) on non‐treatment days. Following completion of pretreatment and training, animals were tested after receiving an ethanol (1.0, 1.5, or 2.0 g/kg), or saline. Tests for experiments II, III, and IV were repeated in the same animals following 12 ethanol‐free days.

Results: Chronic intermittent ethanol exposure during adolescence caused differential weight gain (experiment I). Adolescent rats developed tolerance to ethanol‐induced LORR (experiment II) and metabolic tolerance to ethanol (experiment III). This tolerance was seen after 12 ethanol‐free days. CIE also attenuated ethanol‐induced spatial memory deficits in the MWMT (experiment IV). This effect was not long‐lasting.

Conclusions: Following CIE pretreatment during adolescence, tolerance developed to the hypnotic and cognitive impairing effects of ethanol, along with increased metabolic rate and decreased weight gain. These results further emphasize the ability of CIE to produce a variety of effects during adolescence, some having long‐lasting consequences.
Impairment in Cognitive Functions After Multiple Detoxifications in Alcoholic Inpatients
Authors: Duka Theodora , Townshend Julia M. , Collier Kirsty , Stephens David N.
Published: November 2003

Background: Repeated experience of withdrawal from alcohol results in a kindling‐like process leading to increased likelihood and severity of convulsions during detoxification. The aim of this study was to determine whether repeated withdrawals affect cognitive function.

Methods: We investigated alcoholic patients undergoing detoxification in an inpatient setting, using tasks sensitive to dysfunction of prefrontal areas. The tasks applied were two maze tasks from the Wechsler Intelligence Scale for Children, the color Stroop task, and the vigilance task for adults and the delay task from the Gordon Diagnostic System. Forty‐two abstinent alcoholic patients who were no longer receiving pharmacotherapy for detoxification participated.

Results: Compared to a group of forty‐three social drinkers matched for age, sex, and verbal IQ, the alcoholic patients took more time to complete maze 1 and made more errors in both mazes. Alcoholics made more commission errors and gave fewer correct answers in the vigilance task. No differences were found in the color Stroop task between alcoholic patients and social drinkers. Patients with 2 or more detoxifications were more impaired in the maze 1, in the vigilance task and in the delay task than patients with a single, or no previous detoxification. When patients were reclassified on the basis of the total number of attempts at withdrawing from alcohol (including the medically supervised) only the deficit in the vigilance task was associated with the number of withdrawal attempts. The effects of medically supervised detoxifications on maze 1 and vigilance task were confounded with other factors related to the history of alcoholism, alcohol use, age of starting heavy drinking and years of problem drinking.

Conclusions: Repeated experience of withdrawal from alcohol is thus associated with impaired cognitive function although it appears that for some of these effects, other factors associated with the history of alcoholism might also be involved.
Ethanol Decreases Natural Killer Cell Activation but Only Minimally Affects Anatomical Distribution After Administration of Polyinosinic:Polycytidylic Acid: Role in Resistance to B16F10 Melanoma
Authors: Hébert Pamela , Pruett Stephen B.
Published: November 2003

Background: Natural killer (NK) cells are critical in resistance to B16F10 lung metastases in B6C3F1 mice. Activation of NK cells by polyinosinic:polycytidylic acid (poly I:C; 0.1 mg, intraperitoneally) increases resistance to B16F10 cells. This effect is reduced after administration of ethanol (EtOH; 6 g/kg by oral gavage). The present study was conducted to determine whether decreased resistance is due to alteration of the distribution and/or the activation of NK cells.

Methods: These parameters were measured in the spleen, lungs, and peripheral blood 4 and 12 hr after EtOH and poly I:C administration. For assessing the time after poly I:C administration during which NK cells are important in resistance to B16F10 cells, anti‐NK1.1 antibody was used to deplete NK cells in vivo 48 hr before and 0, 6, 12, and 24 hr after intravenous injection of B16F10 tumor cells.

Results: Depletion of NK cells at any time up to 12 hr after B16F10 administration significantly increased the number of tumor nodules in the lungs, but depletion at 24 hr had a smaller effect. Flow cytometry revealed that there was a small but significant increase in the percentage of NK cells in the lungs at 12 hr, which was not changed by EtOH. Corresponding NK cell lytic function in the lungs was increased significantly at both 4 and 12 hr by poly I:C. However, the increase was not significantly different from the naive control value at 4 hr in mice treated with poly I:C plus EtOH, indicating that EtOH decreased activation of NK cells in the lungs at 4 hr. In the spleen, no treatment significantly altered the percentage of NK cells at 4 or 12 hr. However, poly I:C significantly enhanced lytic function, and this enhancement was suppressed by EtOH (by ∼50%). In the blood, the only significant change in NK cell percentage or lytic activity was an increase in the percentage of NK cells at 12 hr, which was equivalent in the poly I:C and the poly I:C plus EtOH groups.

Conclusions: These results demonstrate that EtOH partially abrogates the poly I:C–induced enhancement of resistance to B16F10 cells and that decreased activation of NK cells in the lungs at a critical time early in the response to poly I:C may contribute to this effect. Other parameters could also contribute, but there was little support for an important role for changes in NK cell distribution.
Methodological and Statistical Considerations in Measuring Alcohol Treatment Effects
Authors: Stout Robert L.
Published: November 2003

This article addresses the design and selection of outcome methods for treatment research from a statistical perspective. Two of the major statistical and methodological issues relevant to the selection of dependent variable(s) are statistical power and social utility. Power is the paramount issue in research design. Power can be improved by measures and methods tailored to the predicted effects of treatment. These effects should vary from treatment to treatment, suggesting that no single outcome measure is suitable for all purposes. In estimating power, researchers should also consider the time‐varying nature of most types of treatment effects. Given these considerations, timeline procedures have advantages because they allow the derivation of measures linked to specific treatment components. They also permit the study of time‐varying effects of variables such as life stress. An analysis on the relationship between drinking and self‐reported stress is presented as an illustration of how advanced statistical methods, in conjunction with carefully gathered data, can provide process data on how treatment can affect individual responses to stressors or other events. From a social utility perspective, however, having research focus on narrower and narrower slices of behavior in the search for power raises questions about the value of this research to clients and society. For dealing with this dilemma, it will be necessary to build a series of studies linking improvement in specific aspects of short‐term outcome to longer term outcome and ultimately benefit to society.
Pin1 modulates the structure and function of human RNA polymerase II
Authors: Xu Yu-Xin , Hirose Yutaka , Zhou Xiao Zhen , Lu Kun Ping , Manley James L.
Published: November 2003

The C-terminal domain of the RNA polymerase (RNAP) II largest subunit (CTD) plays critical roles both in transcription of mRNA precursors and in the processing reactions needed to form mature mRNAs. The CTD undergoes dynamic changes in phosphorylation during the transcription cycle, and this plays a significant role in coordinating its multiple activities. But how these changes themselves are regulated is not well understood. Here we show that the peptidyl-prolyl isomerase Pin1 influences the phosphorylation status of the CTD in vitro by inhibiting the CTD phosphatase FCP1 and stimulating CTD phosphorylation by cdc2/cyclin B. This is reflected in vivo by accumulation of hypophosphorylated RNAP II in pin1-/- cells, and of a novel hyper-hyperphosphorylated form in cells induced to overexpress Pin1. This hyper-hyperphosphorylated form of RNAP II also accumulates in M-phase cells, in a Pin1-dependent manner, and associates specifically with Pin1. Functionally, we find that Pin1 overexpression specifically inhibits ongoing transcription of mRNA precursors in vivo and both transcription and RNAP II-stimulated pre-mRNA splicing in cell extracts. Pin1 thus plays a significant role in regulating RNAP II CTD structure and function.
Pathogen-Responsive Expression of a Putative ATP-Binding Cassette Transporter Gene Conferring Resistance to the Diterpenoid Sclareol Is Regulated by Multiple Defense Signaling Pathways in Arabidopsis
Authors: Campbell Emma J. , Schenk Peer M. , Kazan Kemal , Penninckx Iris A.M.A. , Anderson Jonathan P. , Maclean Don J. , Cammue Bruno P.A. , Ebert Paul R. , Manners John M.
Published: November 2003

The ATP-binding cassette (ABC) transporters are encoded by large gene families in plants. Although these proteins are potentially involved in a number of diverse plant processes, currently, very little is known about their actual functions. In this paper, through a cDNA microarray screening of anonymous cDNA clones from a subtractive library, we identified an Arabidopsis gene (AtPDR12) putatively encoding a member of the pleiotropic drug resistance (PDR) subfamily of ABC transporters. AtPDR12 displayed distinct induction profiles after inoculation of plants with compatible and incompatible fungal pathogens and treatments with salicylic acid, ethylene, or methyl jasmonate. Analysis of AtPDR12 expression in a number of Arabidopsis defense signaling mutants further revealed that salicylic acid accumulation, NPR1 function, and sensitivity to jasmonates and ethylene were all required for pathogen-responsive expression of AtPDR12. Germination assays using seeds from an AtPDR12 insertion line in the presence of sclareol resulted in lower germination rates and much stronger inhibition of root elongation in the AtPDR12 insertion line than in wild-type plants. These results suggest that AtPDR12 may be functionally related to the previously identified ABC transporters SpTUR2 and NpABC1, which transport sclareol. Our data also point to a potential role for terpenoids in the Arabidopsis defensive armory.
Reduced Expression of Aconitase Results in an Enhanced Rate of Photosynthesis and Marked Shifts in Carbon Partitioning in Illuminated Leaves of Wild Species Tomato
Authors: Carrari Fernando , Nunes-Nesi Adriano , Gibon Yves , Lytovchenko Anna , Loureiro Marcelo Ehlers , Fernie Alisdair R.
Published: November 2003

Wild species tomato (Lycopersicon pennellii) plants bearing a genetic lesion in the gene encoding aconitase (Aco-1; aconitate hydratase EC 4.2.1.3) were characterized at molecular and biochemical levels. The genetic basis of this lesion was revealed by cloning the wild-type and mutant alleles. The mutation resulted in lowered expression of the Aco-1 transcript and lowered levels of both cytosolic and mitochondrial aconitase protein and activity. After in silico analysis, we concluded that in the absence of a recognizable target sequence, the best explanation for the dual location of this protein is inefficient targeting. Biochemical analysis of leaves of the Aco-1 accession suggested that they exhibited a restricted flux through the Krebs cycle and reduced levels of Krebs cycle intermediates but were characterized by elevated adenylate levels and an enhanced rate of CO2 assimilation. Furthermore, the analysis of both steady-state metabolite levels and metabolic fluxes revealed that this accession also exhibited elevated rates of photosynthetic Suc synthesis and a corresponding increase in fruit yield. Therefore, we conclude that the Krebs cycle normally competes with the Suc synthetic pathway for carbon but is not essential for the supply of energy to fuel the operation of this pathway.
Changes in Mitochondrial Electron Partitioning in Response to Herbicides Inhibiting Branched-Chain Amino Acid Biosynthesis in Soybean
Authors: Gaston Susana , Ribas-Carbo Miquel , Busquets Silvia , Berry Joseph A. , Zabalza Ana , Royuela Mercedes
Published: November 2003

The adaptation of the respiratory metabolism in roots of soybean (Glycine max L. Merr. cv Ransom) treated with herbicides that inhibit the enzyme acetolactate synthase (ALS) was analyzed. A new gas phase dual-inlet mass spectrometry system for simultaneous measurement of 34O2 to 32O2 and O2 to N2 ratios has been developed. This system is more accurate than previously described systems, allows measurements of much smaller oxygen gradients, and, as a consequence, works with tissues that have lower respiration rates. ALS inhibition caused an increase of the alternative oxidase (AOX) protein and an accumulation of pyruvate. The combination of these two effects is likely to induce the activation of the alternative pathway and its participation in the total respiration. Moreover, the start of the alternative pathway activation and the increase of AOX protein were before the decline in the activity of cytochrome pathway. The possible role of AOX under ALS inhibition is discussed.
p-Chlorophenoxyisobutyric Acid Impairs Auxin Response in Arabidopsis Root
Authors: Oono Yutaka , Ooura Chiharu , Rahman Abidur , Aspuria Evalour T. , Hayashi Ken-ichiro , Tanaka Atsushi , Uchimiya Hirofumi
Published: November 2003

p-Chlorophenoxyisobutyric acid (PCIB) is known as a putative antiauxin and is widely used to inhibit auxin action, although the mechanism of PCIB-mediated inhibition of auxin action is not characterized very well at the molecular level. In the present work, we showed that PCIB inhibited BA::β-glucuronidase (GUS) expression induced by indole-3-acetic acid (IAA), 2,4-dichlorophenoxyacetic acid, and 1-naphthaleneacetic acid. PCIB also inhibited auxin-dependent DR5::GUS expression. RNA hybridization and quantitative reverse transcriptase-polymerase chain reaction analyses suggested that PCIB reduced auxin-induced accumulation of transcripts of Aux/IAA genes. In addition, PCIB relieved the reduction of GUS activity in HS::AXR3NT-GUS transgenic line in which auxin inhibits GUS activity by promoting degradation of the AXR3NT-GUS fusion protein. Physiological analysis revealed that PCIB inhibited lateral root production, gravitropic response of roots, and growth of primary roots. These results suggest that PCIB impairs auxin-signaling pathway by regulating Aux/IAA protein stability and thereby affects the auxin-regulated Arabidopsis root physiology.
Agrobacterium-Mediated Root Transformation Is Inhibited by Mutation of an Arabidopsis Cellulose Synthase-Like Gene
Authors: Zhu Yanmin , Nam Jaesung , Carpita Nicholas C. , Matthysse Ann G. , Gelvin Stanton B.
Published: November 2003

Agrobacterium-mediated plant genetic transformation involves a complex interaction between the bacterium and the host plant. Relatively little is known about the role plant genes and proteins play in this process. We previously identified an Arabidopsis mutant, rat4, that is resistant to Agrobacterium transformation. We show here that rat4 contains a T-DNA insertion into the 3′-untranslated region of the cellulose synthase-like gene CSLA9. CSLA9 transcripts are greatly reduced in the rat4 mutant. Genetic complementation of rat4 with wild-type genomic copies of the CSLA9 gene restores both transformation competence and the wild-type level of CSLA9 transcripts. The CSLA9 promoter shows a distinct pattern of expression in Arabidopsis plants. In particular, the promoter is active in the elongation zone of roots, the root tissue that we previously showed is most susceptible to Agrobacterium-mediated transformation. Disruption of the CSLA9 gene in the rat4 mutant results in reduced numbers and rate of growth of lateral roots and reduced ability of the roots to bind A. tumefaciens cells under certain conditions. No major differences in the linkage structure of the non-cellulosic polysaccharides could be traced to the defective CSLA9 gene.
Evidence for peripheral plasticity in human odour response
Authors: Wang Liwei , Chen Lixin , Jacob Tim
Published: November 2003

Of those people who are anosmic to androstenone, a proportion can acquire sensitivity to it by repeated exposure and even those who are able to smell it can lower their threshold with this treatment. Using olfactory threshold testing, intranasal electrophysiology and EEG we show for the first time that: (1) the subjects' detection threshold is proportional to the amplitude of the olfactory evoked potential (EOG) recorded inside the nose; (2) the EOG amplitude is correlated with the amplitude of the olfactory event‐related potential (OERP) recorded on the scalp; and (3) with repetitive exposure, human subjects acquire a reduced threshold for androstenone and, as they do so, their EOG and OERP increase. These observations support the existence of odourant‐specific plasticity in the peripheral olfactory system.
Yeast Life-Span Extension by Calorie Restriction Is Independent of NAD Fluctuation
Authors: Anderson Rozalyn M. , Latorre-Esteves Magda , Neves Ana Rute , Lavu Siva , Medvedik Oliver , Taylor Christopher , Howitz Konrad T. , Santos Helena , Sinclair David A.
Published: November 2003

Calorie restriction (CR) slows aging in numerous species. In the yeast
Saccharomyces cerevisiae
, this effect requires Sir2, a conserved NAD
+
-dependent deacetylase. We report that CR reduces nuclear NAD
+
levels in vivo. Moreover, the activity of Sir2 and its human homologue SIRT1 are not affected by physiological alterations in the NAD
+
:NADH ratio. These data implicate alternate mechanisms of Sir2 regulation by CR.
The Conformation of the 3′ End of the Minus-Strand DNA Makes Multiple Contributions to Template Switches during Plus-Strand DNA Synthesis of Duck Hepatitis B Virus
Authors: Habig Jeffrey W. , Loeb Daniel D.
Published: November 2003

Two template switches are necessary during plus-strand DNA synthesis of the relaxed circular (RC) form of the hepadnavirus genome. The 3′ end of the minus-strand DNA makes important contributions to both of these template switches. It acts as the donor site for the first template switch, called primer translocation, and subsequently acts as the acceptor site for the second template switch, termed circularization. A small DNA hairpin has been shown to form near the 3′ end of the minus-strand DNA overlapping the direct repeat 1 in avihepadnaviruses. Previously we showed that this hairpin is involved in discriminating between two mutually exclusive pathways for the initiation of plus-strand DNA synthesis. In its absence, the pathway leading to production of duplex linear DNA is favored, whereas primer translocation is favored in its presence, apparently through the inhibition of in situ priming. Circularization involves transfer of the nascent plus strand from the 5′ end of the minus-strand DNA to the 3′ end, where further elongation can lead to production of RC DNA. Using both genetic and biochemical approaches, we now have found that the small DNA hairpin in the duck hepatitis B virus (DHBV) makes a positive contribution to circularization. The contribution appears to be through its impact on the conformation of the acceptor site. We also identified a unique DHBV variant that can synthesize RC DNA well in the absence of the hairpin. The behavior of this variant could serve as a model for understanding the mammalian hepadnaviruses, in which an analogous hairpin does not appear to exist.
Template Switches during Plus-Strand DNA Synthesis of Duck Hepatitis B Virus Are Influenced by the Base Composition of the Minus-Strand Terminal Redundancy
Authors: Habig Jeffrey W. , Loeb Daniel D.
Published: November 2003

Two template switches are necessary during plus-strand DNA synthesis of the relaxed circular (RC) form of the hepadnavirus genome. The 3′ end of the minus-strand DNA makes important contributions to both of these template switches. It acts as the donor site for the first template switch, called primer translocation, and subsequently acts as the acceptor site for the second template switch, termed circularization. Circularization involves transfer of the nascent 3′ end of the plus strand from the 5′ end of the minus-strand DNA to the 3′ end, where further elongation can lead to production of RC DNA. In duck hepatitis B virus (DHBV), a small terminal redundancy (5′r and 3′r) on the ends of the minus-strand DNA has been shown to be important, but not sufficient, for circularization. We investigated what contribution, if any, the base composition of the terminal redundancy made to the circularization process. Using a genetic approach, we found a strong positive correlation between the fraction of A and T residues within the terminal redundancy and the efficiency of the circularization process in those variants. Additionally, we found that the level of in situ priming increases, at the expense of primer translocation, as the fraction of A and T residues in the 3′r decreases. Thus, a terminal redundancy rich in A and T residues is important for both plus-strand template switches in DHBV.
The Human
Polycomb
Group EED Protein Interacts with the Integrase of Human Immunodeficiency Virus Type 1
Authors: Violot Sébastien , Hong Saw See , Rakotobe Dina , Petit Caroline , Gay Bernard , Moreau Karen , Billaud Geneviève , Priet Stéphane , Sire Joséphine , Schwartz Olivier , Mouscadet Jean-François , Boulanger Pierre
Published: November 2003

Human EED, a member of the superfamily of WD-40 repeat proteins and of the
Polycomb
group proteins, has been identified as a cellular partner of the human immunodeficiency virus type 1 (HIV-1) matrix (MA) protein (R. Peytavi et al., J. Biol. Chem. 274:1635-1645, 1999). In the present study, EED was found to interact with HIV-1 integrase (IN) both in vitro and in vivo in yeast. In vitro, data from mutagenesis studies, pull-down assays, and phage biopanning suggested that EED-binding site(s) are located in the C-terminal domain of IN, between residues 212 and 264. In EED, two putative discrete IN-binding sites were mapped to its N-terminal moiety, at a distance from the MA-binding site, but EED-IN interaction also required the integrity of the EED last two WD repeats. EED showed an apparent positive effect on IN-mediated DNA integration reaction in vitro, in a dose-dependent manner. In situ analysis by immunoelectron microscopy (IEM) of cellular distribution of IN and EED in HIV-1-infected cells (HeLa CD4
+
cells or MT4 lymphoid cells) showed that IN and EED colocalized in the nucleus and near nuclear pores, with maximum colocalization events occurring at 6 h postinfection (p.i.). Triple colocalizations of IN, EED, and MA were also observed in the nucleoplasm of infected cells at 6 h p.i., suggesting the ocurrence of multiprotein complexes involving these three proteins at early steps of the HIV-1 virus life cycle. Such IEM patterns were not observed with a noninfectious, envelope deletion mutant of HIV-1.