bluuebunny/crossref_metadata_2025 huggingface dataset:
92 INTER-GENERIC MARBLED CAT AND FLAT-HEADED CAT CLONED BLASTOCYSTS GENERATED FROM DOMESTIC CAT AND RABBIT OOCYTES
Authors: Thongphakdee A. , Numchaisrika P. , Ratanakorn P. , Kamolnorranath S. , Chatdarong K. , Techakumphu M.
Published: December 2006

Marbled cats (Pardofelis marmorata; MC) and flat-headed cats (Prionailurus planiceps; FC), the endangered, small wild cats of southeast Asia, are currently at risk of becoming extinct in Thailand. Producing embryos by inter-generic somatic cell nuclear transfer (SCNT) is a way to preserve their genetics, as well as to understand their embryo chronology. The aim of the study was to investigate the development of MC and FC embryos produced from domestic cat (DC) and rabbit (RB) oocytes by SCNT. Intra-species cloned DC served as control. The MC-, FC-, DC-DC, and FC-RB (donor fibroblast cell–recipient oocyte) couplets were prepared as previously described (Thongphakdee et al. 2006 Reprod. Dom. Anim. 41, 219–226), but MC-, FC-, and DC-DC couplets were fused by inducing 3 direct current pulses of 2.4 kV cm-1, for 50 �s, and subsequently activated in synthetic oviduct fluid (SOF) medium, supplemented with 10% fetal bovine serum (FBS), 10 �g mL-1 cycloheximide, and 5 �g mL-1 cytochalasin B, for 4 h. Activated couplets were cultured in SOF medium supplemented with 5% FBS, at 38.5�C, under 5% CO2 in air, for 5–7 days. Differences in the percentages of fusion and embryo development to a particular stage between species were determined by chi-square analysis. The fusion efficiency of DC-DC couplets (38%; 36/94) was significantly lower than those of MC-DC (74%; 60/81), FC-DC (79%; 60/76), and FC-RB (78%; 47/60) couplets (P < 0.05). No differences in the cleavage rates of MC-DC, FC-DC, FC-RB, and DC-DC fused couplets were found (93%, 97%, 87%, and 89%, respectively). The FC-DC couplets yielded a greater percentage of morulae (53%) than did MC-DC (23%), FC-RB (25.5%), and DC-DC (11%) couplets (P < 0.05). However, the number of couplets that achieved blastocyst expansion did not significantly differ (MC-DC: 5%, FC-DC: 8.3%, FC-RB: 8.5%, and DC-DC: 8.5%). These results demonstrate that MC and FC embryos are successfully produced with reasonable blastocyst formation rates by inter-generic SCNT.
This study was supported by the Zoological Park Organization under the Royal Patronage of H.M. the King, Reproductive Biotechnology Research Unit, Chulalongkorn University, and Royal Golden Jubilee, PhD program of Thailand Research Fund.
96 TRICHOSTATIN A IMPROVED THE QUALITY OF RABBIT NUCLEAR TRANSFER EMBRYOS
Authors: Xu J. , Sung L.-Y. , Zhang J. , Tian X. , Chen Y. E. , Yang X. , Du F.
Published: December 2006

Nuclear reprogramming is dependent upon a number of factors, including chromatin organization and modification. Trychostatin A (TSA), a histone deacetylase inhibitor, was used to increase histone acetylation and to improve reprogrammability in both cattle and mice. The objective of the study was to determine whether TSA could improve the pre-implantational development potential of rabbit nuclear transplant (NT) embryos. Rabbit oocytes were flushed from the oviducts of superovulated donors treated with the regime of FSH and hCG. Cumulus cells were then denuded from the oocytes by incubation in 0.5% hyaluronidase and pipetting. Oocyte enucleation was conducted in 10% FBS M199 and confirmed under fluorescence microscopy. Cumulus cells were prepared as nuclear donors for NT; a donor cell with the diameter approximately 15–19 µm was transferred into the perivitelline space of an enucleated oocyte, and subsequently fused with the oocyte recipient by application of 3 direct current pulses at 3.2 kV cm−1 for a duration of 20 µs/pulse. Fused embryos were activated by the same electrical stimulation regime described above, and subsequently cultured in M199 + 10% FBS containing 2.0 mM 6-dimethylaminopurine (DMAP) and 5 µg mL−1 cycloheximide for 1 h. Rabbit NT embryos were cultured in 5 nM TSA-2.5% FBS-B2 medium for 10 h before being transferred into regular medium (FBS-B2). The TSA-treated embryos (5 nM vs. 0 nM) were cultured in 400 µL FBS-B2 medium for 5 days in 5% CO2 in a humidified atmosphere at 38.5°C (initiation of activation = Day 0). NT embryo development to cleaved (2 to 4 cell), morula, and blastocyst stages was evaluated on Day 2, Day 3, and Day 5, respectively. The selected NT blastocysts were counted for cell numbers following Hoechst 33342 epifluorescenin staining. The results (Table 1) showed that there was no difference on pre-implantational development of cloned embryos between TSA-added and control groups (P > 0.05). However, a significantly higher cell number per NT blastocyst was found in the TSA-added group (357 vs. 113; P < 0.05). This indicated that the blastocyst quality in NT embryos was improved with the addition of TSA by increasing histone acetylation activity. The developmental potential of TSA-treated NT embryos to term is under investigation.

Table 1.Effects of TSA on the pre-implantational development of cloned rabbit embryos

This work was supported by NIH/NCRR-SBIR grant: 1R43RR020261-01.
99 AMOUNT OF TELOMERIC DNA IN GROWING CLONED CATTLE, THEIR PROGENY, AND THEIR ORGANS
Authors: Yang B. C. , Im G. S. , Kim Y. H. , Kim D. H. , Bae S. H. , Ko Y. G. , Seong H. H. , Shon S. H. , Yang B. S.
Published: December 2006

Telomeres are specialized nucleoprotein complexes at the termini of linear chromosomes that are composed of TTAGGG sequences in vertebrates. Telomere lengths in animals vary with species, age, tissue types, environment, and cloning. The experiment conducted emphasized the amount of telomeric DNA in the lymphocytes and organs of growing cloned cattle and their second and third generations. Using somatic cell nuclear transfer (SCNT), 16 cloned (Generation, clone G1) Korean Native cows were obtained from ear skin fibroblasts and 2 cloned bulls from fetal fibroblasts. In addition, 3 females and 2 males (clone G2) were produced from each cloned cow by artificial insemination (AI). A third generation calf (clone G3) was derived from clone G2 by AI. The lymphocytes of all cloned cattle (G1), their offspring (G2), and age-matched controls were examined 3 times at 6-month intervals whereas G3 was examined only once. The amount of telomeric DNA was analyzed by quantitative fluorescence in situ hybridization (Q-FISH) with a human telomeric DNA repeat probe. A minimum of 100 interphase nuclei from each set of harvests was studied to determine the mean and medium percentages of telomeric DNA using MetaMorph Imaging System (Universal Imaging Co, West Chester, PA, USA). The amounts of telomeric DNA in cloned cattle from both ear skin fibroblasts (female, n = 16) and fetal fibroblasts (male, n = 2) were less than those of age-matched controls (P < 0.01). Additionally, irrespective of gender, the telomeres in the clone G2 and G3 calves were lower than in controls (n = 6; P < 0.05). Furthermore, in the cloned cattle, the amount of telomeric DNA was drastically less than that of control animals during growth. Moreover, we examined the internal organs and tissues of a cloned cow at 30 months. The telomeres of leukocytes, cerebrum, spleen, cerebellum, hindbrain, and lung were a little smaller, whereas those of the liver, pituitary, kidney, and heart were slightly larger, than those of an age-matched cow. The results showed a remarkable difference in the amount of telomeric DNA between SCNT cloned cattle and normal cattle. Although the organs and tissues were not correlated, the amount of telomeres rapidly decreased with growth in cloned cattle. Conclusively, the telomeres of a cloned animal and its calves were significantly shorter than those of control cattle, and the short telomeres in calves could be inherited by progeny from their cloned mother.
Gain-of-function mutations in complement factor B are associated with atypical hemolytic uremic syndrome
Authors: de Jorge Elena Goicoechea , Harris Claire L. , Esparza-Gordillo Jorge , Carreras Luis , Arranz Elena Aller , Garrido Cynthia Abarrategui , López-Trascasa Margarita , Sánchez-Corral Pilar , Morgan B. Paul , de Córdoba Santiago Rodríguez
Published: December 2006

Hemolytic uremic syndrome (HUS) is an important cause of acute renal failure in children. Mutations in one or more genes encoding complement-regulatory proteins have been reported in approximately one-third of nondiarrheal, atypical HUS (aHUS) patients, suggesting a defect in the protection of cell surfaces against complement activation in susceptible individuals. Here, we identified a subgroup of aHUS patients showing persistent activation of the complement alternative pathway and found within this subgroup two families with mutations in the gene encoding factor B (BF), a zymogen that carries the catalytic site of the complement alternative pathway convertase (C3bBb). Functional analyses demonstrated that F286L and K323E aHUS-associatedBFmutations are gain-of-function mutations that result in enhanced formation of the C3bBb convertase or increased resistance to inactivation by complement regulators. These data expand our understanding of the genetic factors conferring predisposition to aHUS, demonstrate the critical role of the alternative complement pathway in the pathogenesis of aHUS, and provide support for the use of complement-inhibition therapies to prevent or reduce tissue damage caused by dysregulated complement activation.
Positive affect increases the breadth of attentional selection
Authors: Rowe G. , Hirsh J. B. , Anderson A. K.
Published: December 2006

The present study examined the thesis that positive affect may serve to broaden the scope of attentional filters, reducing their selectivity. The effect of positive mood states was measured in two different cognitive domains: semantic search (remote associates task) and visual selective attention (Eriksen flanker task). In the conceptual domain, positive affect enhanced access to remote associates, suggesting an increase in the scope of semantic access. In the visuospatial domain, positive affect impaired visual selective attention by increasing processing of spatially adjacent flanking distractors, suggesting an increase in the scope of visuospatial attention. During positive states, individual differences in enhanced semantic access were correlated with the degree of impaired visual selective attention. These findings demonstrate that positive states, by loosening the reins on inhibitory control, result in a fundamental change in the breadth of attentional allocation to both external visual and internal conceptual space.
Human Sco1 functional studies and pathological implications of the P174L mutant
Authors: Banci Lucia , Bertini Ivano , Ciofi-Baffoni Simone , Leontari Iliana , Martinelli Manuele , Palumaa Peep , Sillard Rannar , Wang Shenlin
Published: December 2006

The pathogenic mutant (P174L) of human Sco1 produces respiratory chain deficiency associated with cytochrome
c
oxidase (CcO) assembly defects. The solution structure of the mutant in its Cu(I) form shows that Leu-174 prevents the formation of a well packed hydrophobic region around the metal-binding site and causes a reduction of the affinity of copper(I) for the protein.
K
D
values for Cu(I)WT-HSco1 and Cu(I)P174L-HSco1 are ≈10
−17
and ≈10
−13
, respectively. The reduction potentials of the two apo proteins are similar, but slower reduction/oxidation rates are found for the mutant with respect to the WT. The mitochondrial metallochaperone in the partially oxidized Cu
1
(I)Cox17
2S-S
form, at variance with the fully reduced Cu
4
(I)Cox17, interacts transiently with both WT-HSco1 and the mutant, forming the Cox17/Cu(I)/HSco1 complex, but copper is efficiently transferred only in the case of WT protein. Cu
1
(I)Cox17
2S-S
indeed has an affinity for copper(I) (
K
D
≈ 10
−15
) higher than that of the P174L-HSco1 mutant but lower than that of WT-HSco1. We propose that HSco1 mutation, altering the structure around the metal-binding site, affects both copper(I) binding and redox properties of the protein, thus impairing the efficiency of copper transfer to CcO. The pathogenic mutation therefore could (
i
) lessen the Sco1 affinity for copper(I) and hence copper supply for CcO or (
ii
) decrease the efficiency of reduction of CcO thiols involved in copper binding, or both effects could be produced by the mutation.
RecA-independent recombination is efficient but limited by exonucleases
Authors: Dutra Bethany E. , Sutera Vincent A. , Lovett Susan T.
Published: December 2006

Genetic recombination in bacteria is facilitated by the RecA strand transfer protein and strongly depends on the homology between interacting sequences. RecA-independent recombination is detectable but occurs at extremely low frequencies and is less responsive to the extent of homology. In this article, we show that RecA-independent recombination in
Escherichia coli
is depressed by the redundant action of single-strand exonucleases. In the absence of multiple single-strand exonucleases, the efficiency of RecA-independent recombination events, involving either gene conversion or crossing-over, is markedly increased to levels rivaling RecA-dependent events. This finding suggests that RecA-independent recombination is not intrinsically inefficient but is limited by single-strand DNA substrate availability. Crossing-over is inhibited by exonucleases ExoI, ExoVII, ExoX, and RecJ, whereas only ExoI and RecJ abort gene-conversion events. In ExoI
−
RecJ
−
strains, gene conversion can be accomplished by transformation of short single-strand DNA oligonucleotides and is more efficient when the oligonucleotide is complementary to the lagging-strand replication template. We propose that
E. coli
encodes an unknown mechanism for RecA-independent recombination (independent of prophage recombination systems) that is targeted to replication forks. The potential of RecA-independent recombination to mediate exchange at short homologies suggests that it may contribute significantly to genomic change in bacteria, especially in species with reduced cellular exonuclease activity or those that encode DNA protection factors.
cAMP activation of PKA defines an ancient signaling mechanism
Authors: Das Rahul , Esposito Veronica , Abu-Abed Mona , Anand Ganesh S. , Taylor Susan S. , Melacini Giuseppe
Published: December 2006

cAMP and the cAMP binding domain (CBD) constitute a ubiquitous regulatory switch that translates an extracellular signal into a biological response. The CBD contains α- and β-subdomains with cAMP binding to a phosphate binding cassette (PBC) in the β-sandwich. The major receptors for cAMP in mammalian cells are the regulatory subunits (R-subunits) of PKA where cAMP and the catalytic subunit compete for the same CBD. The R-subunits inhibit kinase activity, whereas cAMP releases that inhibition. Here, we use NMR to map at residue resolution the cAMP-dependent interaction network of the CBD-A domain of isoform Iα of the R-subunit of PKA. Based on H/D, H/H, and N
z
exchange data, we propose a molecular model for the allosteric regulation of PKA by cAMP. According to our model, cAMP binding causes long-range perturbations that propagate well beyond the immediate surroundings of the PBC and involve two key relay sites located at the C terminus of β
2
(I163) and N terminus of β
3
(D170). The I163 site functions as one of the key triggers of global unfolding, whereas the D170 locus acts as an electrostatic switch that mediates the communication between the PBC and the B-helix. Removal of cAMP not only disrupts the cap for the B′ helix within the PBC, but also breaks the circuitry of cooperative interactions stemming from the PBC, thereby uncoupling the α- and β-subdomains. The proposed model defines a signaling mechanism, conserved in every genome, where allosteric binding of a small ligand disrupts a large protein–protein interface.
NMDA-dependent facilitation of corticostriatal plasticity by the amygdala
Authors: Popescu Andrei T. , Saghyan Aleksandr A. , Paré Denis
Published: December 2006

Emotions generally improve memory, and the basolateral amygdala (BLA) is believed to mediate this effect. After emotional arousal, BLA neurons increase their firing rate, facilitating memory consolidation in BLA targets. The enhancing effects of BLA activity extend to various types of memories, including motor learning, which is thought to involve activity-dependent plasticity at corticostriatal synapses. However, the underlying mechanisms are unknown. Here we show that the NMDA-to-AMPA (α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid) ratio is nearly twice as high at BLA as compared with cortical synapses onto principal striatal neurons and that activation of BLA inputs greatly facilitates long-term potentiation induction at corticostriatal synapses. This facilitation was NMDA-dependent, but it occurred even when BLA and cortical stimuli were 0.5 s apart during long-term potentiation induction. Overall, these results suggest that BLA activity opens long time windows during which the induction of corticostriatal plasticity is facilitated.
A quantitative study of bioenergetics in skeletal muscle lacking carbonic anhydrase III using
31
P magnetic resonance spectroscopy
Authors: Liu M. , Walter G. A. , Pathare N. C. , Forster R. E. , Vandenborne K.
Published: December 2006

Oxidative slow skeletal muscle contains carbonic anhydrase III in high concentration, but its primary function remains unknown. To determine whether its lack handicaps energy metabolism and/or acid elimination, we measured the intracellular pH and energy phosphates by
31
P magnetic resonance spectroscopy in hind limb muscles of wild-type and CA III knockout mice during and after ischemia and intense exercise (electrical stimulation). Thirty minutes of ischemia caused phosphocreatine (PCr) to fall and P
i
to rise while pH and ATP remained constant in both strains of mice. PCr and P
i
kinetics during ischemia and recovery were not significantly different between the two genotypes. From this we conclude that under neutral pH conditions resting muscle anaerobic metabolism, the rate of the creatine kinase reaction, intracellular buffering of protons, and phosphorylation of creatine by mitochondrial oxygen metabolism are not influenced by the lack of CA III. Two minutes of intense stimulation of the mouse gastrocnemius caused PCr, ATP, and pH to fall and ADP and P
i
to rise, and these changes, with the exception of ATP, were all significantly larger in the CA III knockouts. The rate of return of pH and ADP to control values was the same in wild-type and mutant mice, but in the mutants PCr and P
i
recovery were delayed in the first minute after stimulation. Because the tension decrease during fatigue is known to be the same in the two genotypes, we conclude that a lack of CA III impairs mitochondrial ATP synthesis.
The power stroke of myosin VI and the basis of reverse directionality
Authors: Bryant Zev , Altman David , Spudich James A.
Published: December 2006

Myosin VI supports movement toward the (−) end of actin filaments, despite sharing extensive sequence and structural homology with (+)-end-directed myosins. A class-specific stretch of amino acids inserted between the converter domain and the lever arm was proposed to provide the structural basis of directionality reversal. Indeed, the unique insert mediates a 120° redirection of the lever arm in a crystal structure of the presumed poststroke conformation of myosin VI [Ménétrey J, Bahloul A, Wells AL, Yengo CM, Morris CA, Sweeney HL, Houdusse A (2005)
Nature
435:779–785]. However, this redirection alone is insufficient to account for the large (−)-end-directed stroke of a monomeric myosin VI construct. The underlying motion of the myosin VI converter domain must therefore differ substantially from the power stroke of (+)-end-directed myosins. To experimentally map out the motion of the converter domain and lever arm, we have generated a series of truncated myosin VI constructs and characterized the size and direction of the power stroke for each construct using dual-labeled gliding filament assays and optical trapping. Motors truncated near the end of the converter domain generate (+)-end-directed motion, whereas longer constructs move toward the (−) end. Our results directly demonstrate that the unique insert is required for directionality reversal, ruling out a large class of models in which the converter domain moves toward the (−) end. We suggest that the lever arm rotates ≈180° between pre- and poststroke conformations.
Symptom Severity, Alcohol Craving, and Age of Trauma Onset in Childhood and Adolescent Trauma Survivors with Comorbid Alcohol Dependence and Posttraumatic Stress Disorder
Authors: Schumacher Julie A. , Coffey Scott F. , Stasiewicz Paul R.
Published: December 2006

Posttraumatic stress disorder (PTSD) and alcohol dependence (AD) are frequently comorbid disorders. Given evidence that childhood traumas may be associated with broader, more severe psychological sequelae than later traumas, the present study examined whether the association between alcohol and trauma symptomatology is more pronounced among individuals with earlier trauma onsets in a sample of 42 childhood and adolescent trauma survivors diagnosed with comorbid AD‐PTSD. As predicted, individuals reporting childhood traumas reported greater severity of trauma and alcohol symptoms and greater alcohol craving. These results suggest that individuals with childhood trauma histories may be particularly vulnerable to relapse following AD treatment.
What Factors Are Associated with High‐Frequency Drug Treatment Use among a Racially and Ethnically Diverse Population of Injection Drug Users?
Authors: Chassler Deborah , Lundgren Lena , Lonsdale Joya
Published: December 2006

This study explored the frequency of drug treatment utilization by 36,081 injection drug users (IDUs) in Massachusetts, 1996–2002. A number of multiple and logistic regression analyses examined the relationship between demographic characteristics, parental status, level of and type of drug use, history of mental health treatment use, types of drug treatment entered, and the number of times an IDU had entered drug treatment for the seven‐year time period. Homelessness, using heroin as the primary drug of choice, and health insurance status were all associated with number of treatments entered. Logistic regression analysis identified that health insurance was a key factor associated with more frequent treatment: those with private health insurance were ten times more likely to be in the 90th percentile (12–107 entries) with respect to number of treatment entries.
Maternal Cigarette Smoking during Pregnancy and Child Aggressive Behavior
Authors: Brook David W. , Zhang Chenshu , Rosenberg Gary , Brook Judith S.
Published: December 2006

This study's objective was to examine the association between maternal smoking during pregnancy and childhood aggressive behavior in African‐American and Puerto Rican children, as well as the relationship between maternal unconventional behavior, low maternal affection, and offspring aggression. Participants consisted of African‐American and Puerto Rican children (n = 203; mean age = 8.6, SD = 0.87) and their mothers living in an inner city community. An interview consisting of a structured questionnaire was administered to the mothers and their children. Scales with adequate psychometric properties were adapted from previous validated measures. They included maternal smoking during pregnancy, maternal education, unconventionality, and warmth. Controlling for demographic factors, maternal unconventional behavior, and low maternal warmth, maternal smoking during pregnancy was associated with having offspring who were aggressive. Maternal unconventionality and warmth were independently related to childhood aggression. Although causal limitations are noted, it may be that a decrease in smoking during pregnancy is associated with a reduction in aggression in the offspring.
Association between Opium Abuse and Comorbidity in Diabetic Men
Authors: Shiri Rahman , Hassani Kobra Falah , Ansari Mostafa
Published: December 2006

The aims of this study were to determine the prevalence of opium abuse in diabetic men and to investigate its association with comorbidity. The study population was comprised of 312 consecutive diabetic men aged 20 years or older residing in the study area in 2005. The prevalence of self‐reported opium abuse was 11.2%. Opium use was associated with low socioeconomic status, smoking, tea consumption, and a higher prevalence of erectile dysfunction (ED) and severe depression. The prevalence of severe depression was 22.8% among 35 men who used opium and 13.4% among 277 who did not use it. The prevalence of moderate or severe ED was 85.7% among opium users and 66.1% among non‐users. The risk of ED was two times (95% CI 1.0–7.4) higher in opium users compared with nonusers.
Adolescent Self‐Selection of Service Formats: Implications for Secondary Interventions Targeting Alcohol Use
Authors: D'Amico Elizabeth J. , Anderson Kristen G. , Metrik Jane , Frissell Kevin C. , Ellingstad Timothy , Brown Sandra A.
Published: December 2006

A novel motivational enhancement based secondary alcohol intervention has been shown to increase quit attempts for youth with heavier alcohol use histories (Brown et al., 2005). In the present study, we examined rates of self‐selection into the three formats of this alcohol intervention: group, individual, and website; and examined differences between intervention participants and the general school population and across the three formats. Over four years, students at four schools were surveyed (n—6000) and were provided the opportunity to participate in Project Options (PO). Youth who were selected into PO (n—1147) were younger and more likely to identify as African American or Multiple/ Other ethnicity than the school populations. More teens in PO reported lifetime alcohol use (65% vs. 60%); however, the school population reported more current (past 30 day) drinking. Boys were more likely to utilize the group format and minority youth were more likely to select the individual format. Findings highlight the utility of multiple intervention formats to engage youth in early intervention for alcohol problems.
Adolescents with Co‐Occurring Substance Use and Mental Conditions in a Private Managed Care Health Plan: Prevalence, Patient Characteristics and Treatment Initiation and Engagement
Authors: Chi Felicia W. , Sterling Stacy , Weisner Constance
Published: December 2006

This study examined the prevalence, patient characteristics, and treatment initiation and engagement of adolescents with co‐occurring substance use (SU) and serious mental health (MH) diagnoses in a private, managed care health plan. We identified 2,005 adolescents aged 12–17, who received both SU and MH diagnoses within a 1‐year window between 1/1/2000 and 12/31/2002; 57% were girls. Gender variations were found in diagnoses received and point of identification. Being dually diagnosed in specialty departments (rather than Primary Care and Emergency) and receiving both diagnoses within a shorter time period were associated with treatment initiation and engagement.
Cognitive Behavioral Treatment for Suicidal Alcohol Abusing Adolescents: Development and Pilot Testing
Authors: Esposito‐Smythers Christianne , Spirito Anthony , Uth Rebecca , LaChance Heather
Published: December 2006

The purpose of this study was to pilot a cognitive behavioral treatment protocol for adolescents with co‐occurring alcohol use disorder and suicidality, examine its association with symptomatic improvement, and determine its feasibility and acceptability. Treatment consisted of a 6 month acute treatment phase, 3 month maintenance phase, and a 3 month booster phase, as well as case management services. Participants were also permitted to receive concurrent pharmacotherapy. Five of six families completed the protocol. Measures of alcohol use and suicidality were collected at intake, end of acute treatment, and post‐treatment. Decreases in alcohol use and suicidal ideation were reported for all participants. Two of the five participants, both with a prior history of suicide attempts, re‐attempted during the course of the protocol. Both were maintained in the study and improved over the subsequent portion of the treatment. A high retention rate, strong therapeutic alliance ratings, and low perceived treatment obstacles provide support for the feasibility and acceptability of this intervention. Preliminary results suggest that integrated outpatient cognitive behavioral treatment for alcohol abusing suicidal adolescents is feasible, acceptable, and associated with symptomatic improvement.
Comparative Efficacy of Family and Group Treatment for Adolescent Substance Abuse
Authors: Smith Douglas C. , Hall James A. , Williams Julie K. , An Hyonggin , Gotman Nathan
Published: December 2006

Due to the continuing prevalence of adolescent substance abuse, promising treatment models need to be developed and evaluated. Thus, the purpose of this study was to evaluate the efficacy of two promising models, Strengths Oriented Family Therapy (SOFT) and The Seven Challenges® (7C). Adolescents who qualified for outpatient treatment and agreed to participate in our study were randomly assigned to one of the two treatments and assessed at 3 and 6‐months following baseline. Using a two‐part, random‐effects model, we examined the odds of achieving abstinence or full symptom remission between treatments and over time. For those not achieving full abstinence or full problem remission, we investigated whether frequency of use or symptom severity were reduced at follow‐up. Participants in both SOFT and 7C demonstrated significant reductions in substance use and related problems, but treatments did not differ at 3 and 6 months following baseline. Overall, treatment services were delivered as planned. Both SOFT and 7C were efficacious with adolescents who abuse substances, as participants in both conditions were significantly more likely to be in symptom remission or abstinent at follow‐up interviews versus at baseline. Replication studies are needed that address this study's limitations.
Employment and Adolescent Alcohol and Drug Treatment and Recovery: An Exploratory Study
Authors: Godley Susan H. , Passetti Lora L. , White Michelle K.
Published: December 2006

Studies of adolescents in the general population show that most high school students are employed and that there is a positive linear relationship between hours worked and increases in alcohol and other drug use. Mixed methods are used to examine the relationship of employment for adolescents who are in outpatient substance abuse treatment to their use, treatment experiences, and recovery. Several theories offered to explain the relationship between adolescent employment and substance use were examined. Most adolescents were employed, often during treatment, with increasing numbers reporting employment over the year‐long follow‐up period. Adolescents reported frequent alcohol and other drug use after work and with coworkers and indicated that income from work often was used to purchase alcohol and other drugs. Most parents did not provide monitoring of work‐related income. Balancing employment and treatment was often logistically difficult and stressful for the adolescents. Employment situations are a critical aspect of adolescents’ recovery environment, and more research is needed to learn how to create support for recovery in this aspect of an adolescent's life.
Small Heat-Shock Proteins Select ΔF508-CFTR for Endoplasmic Reticulum-associated Degradation
Authors: Ahner Annette , Nakatsukasa Kunio , Zhang Hui , Frizzell Raymond A. , Brodsky Jeffrey L.
Published: December 2006

Secreted proteins that fail to achieve their native conformations, such as cystic fibrosis transmembrane conductance regulator (CFTR) and particularly the ΔF508-CFTR variant can be selected for endoplasmic reticulum (ER)-associated degradation (ERAD) by molecular chaperones. Because the message corresponding to HSP26, which encodes a small heat-shock protein (sHsp) in yeast was up-regulated in response to CFTR expression, we examined the impact of sHsps on ERAD. First, we observed that CFTR was completely stabilized in cells lacking two partially redundant sHsps, Hsp26p and Hsp42p. Interestingly, the ERAD of a soluble and a related integral membrane protein were unaffected in yeast deleted for the genes encoding these sHsps, and CFTR polyubiquitination was also unaltered, suggesting that Hsp26p/Hsp42p are not essential for polyubiquitination. Next, we discovered that ΔF508-CFTR degradation was enhanced when a mammalian sHsp, αA-crystallin, was overexpressed in human embryonic kidney 293 cells, but wild-type CFTR biogenesis was unchanged. Because αA-crystallin interacted preferentially with ΔF508-CFTR and because purified αA-crystallin suppressed the aggregation of the first nucleotide-binding domain of CFTR, we suggest that sHsps maintain the solubility of ΔF508-CFTR during the ERAD of this polypeptide.
Mitogen-activated Protein Kinase Kinase 1-dependent Golgi Unlinking Occurs in G2Phase and Promotes the G2/M Cell Cycle Transition
Authors: Feinstein Timothy N. , Linstedt Adam D.
Published: December 2006

Two controversies have emerged regarding the signaling pathways that regulate Golgi disassembly at the G2/M cell cycle transition. The first controversy concerns the role of mitogen-activated protein kinase activator mitogen-activated protein kinase kinase (MEK)1, and the second controversy concerns the participation of Golgi structure in a novel cell cycle “checkpoint.” A potential simultaneous resolution is suggested by the hypothesis that MEK1 triggers Golgi unlinking in late G2to control G2/M kinetics. Here, we show that inhibition of MEK1 by RNA interference or by using the MEK1/2-specific inhibitor U0126 delayed the passage of synchronized HeLa cells into M phase. The MEK1 requirement for normal mitotic entry was abrogated if Golgi proteins were dispersed before M phase by treatment of cells with brefeldin A or if GRASP65, which links Golgi stacks into a ribbon network, was depleted. Imaging revealed that unlinking of the Golgi apparatus begins before M phase, is independent of cyclin-dependent kinase 1 activation, and requires MEK signaling. Furthermore, expression of the GRASP family member GRASP55 after alanine substitution of its MEK1-dependent mitotic phosphorylation sites inhibited both late G2Golgi unlinking and the G2/M transition. Thus, MEK1 plays an in vivo role in Golgi reorganization, which regulates cell cycle progression.
Characterization of Multiple Multivesicular Body Sorting Determinants within Sna3: A Role for the Ubiquitin Ligase Rsp5
Authors: Oestreich Andrea J. , Aboian Mariam , Lee Jacqueline , Azmi Ishara , Payne Johanna , Issaka Rachel , Davies Brian A. , Katzmann David J.
Published: December 2006

A subset of proteins that transit the endosomal system are directed into the intralumenal vesicles of multivesicular bodies (MVBs). MVB formation is critical for a variety of cellular functions including receptor down-regulation, viral budding, antigen presentation, and the generation of lysosome-related organelles. Entry of transmembrane proteins into the intralumenal vesicles of a MVB is a highly regulated process that is positively modulated by covalent modification of cargoes with ubiquitin. To identify additional MVB sorting signals, we examined the previously described ubiquitination-independent MVB cargo Sna3. Although Sna3 ubiquitination is not essential, Sna3 MVB sorting is positively modulated by its ubiquitination. Examination of MVB sorting determinants within a form of Sna3 lacking all lysine residues identified two critical regions: an amino-terminal tyrosine-containing region and a carboxyl-terminal PPAY motif. This PPAY motif interacts with the WW domains of the ubiquitin ligase Rsp5, and mutations in either the WW or, surprisingly, the HECT domains of Rsp5 negatively impacted MVB targeting of lysine-minus Sna3. These data indicate that Rsp5 function is required for MVB targeting of Sna3 in a capacity beyond cargo ubiquitination. These results uncover a series of determinants impacting Sna3 MVB sorting, including unexpected roles for Rsp5.
Trypanosoma bruceiARF1 Plays a Central Role in Endocytosis and Golgi–Lysosome Trafficking
Authors: Price Helen P. , Stark Meg , Smith Deborah F.
Published: December 2006

The ADP ribosylation factor (Arf)1 orthologue in the divergent eukaryote Trypanosoma brucei (Tb) shares characteristics with both Arf1 and Arf6 and has a vital role in intracellular protein trafficking. TbARF1 is Golgi localized in trypanosomes but associates with the plasma membrane when expressed in human cells. Depletion of TbARF1 by RNA interference causes a major decrease in endocytosis, which correlates with Rab5 dissociation from early endosomes. Although the Golgi remains intact, parasites display enlarged flagellar pockets and intracellular flagella. An increase in active GTP-bound TbARF1 in bloodstream parasites is rapidly lethal, correlating with a defect in Golgi-to-lysosome transport. We conclude that the essential Golgi-localizing T. brucei ARF1 has a primary role in the maintenance of both post-Golgi transport and endocytosis and that it is significantly divergent from other characterized ARFs.
Np95 Is Implicated in Pericentromeric Heterochromatin Replication and in Major Satellite Silencing
Authors: Papait Roberto , Pistore Christian , Negri Diego , Pecoraro Daniela , Cantarini Lisa , Bonapace Ian Marc
Published: December 2006

Heterochromatin plays an important role in transcriptional repression, for the correct segregation of chromosomes and in the maintenance of genome stability. Pericentric heterochromatin (PH) replication and formation have been proposed to occur in the pericentric heterochromatin duplication body (pHDB). A central question is how the underacetylated state of heterochromatic histone H4 tail is established and controlled, because it is a key event during PH replication and is essential to maintain the compacted and silenced state of these regions. Np95 is a cell cycle regulated and is a nuclear histone-binding protein that also recruits HDAC-1 to target promoters. It is essential for S phase and for embryonic formation and is implicated in chromosome stability. Here we show that Np95 is part of the pHDB, and its functional ablation causes a strong reduction in PH replication. Depletion of Np95 also causes a hyperacetylation of lysines 8, 12, and 16 of heterochromatin histone H4 and an increase of pericentromeric major satellite transcription, whose RNAs are key players for heterochromatin formation. We propose that Np95 is a new relevant protein involved in heterochromatin replication and formation.
DNA methylation regulates long-range gene silencing of an X-linked homeobox gene cluster in a lineage-specific manner
Authors: Oda Masaaki , Yamagiwa Akiko , Yamamoto Shinji , Nakayama Takao , Tsumura Akiko , Sasaki Hiroshi , Nakao Kazuki , Li En , Okano Masaki
Published: December 2006

DNA methylation is a major epigenetic mechanism that has been suggested to control developmental gene regulation during embryogenesis, but its regulatory mechanisms remain unclear. In this report, we show that CpG islands associated with the X-linked homeobox gene cluster Rhox, which is highly expressed in the extraembryonic trophectoderm, are differentially methylated in a stage- and lineage-specific manner during the post-implantation development of mice. Inactivation of both Dnmt3a and Dnmt3b, DNA methyltransferases essential for the initiation of de novo DNA methylation, abolished the establishment of DNA methylation and the silencing of Rhox cluster genes in the embryo proper. The Dnmt3-dependent CpG-island methylation at the Rhox locus extended for a large genomic region (∼1 Mb) containing the Rhox cluster and surrounding genes. Complementation experiments using embryonic stem (ES) cells deficient in the DNA methyltransferases suggested that the CpG-island methylation by Dnmt3a and Dnmt3b was restricted within this large genomic region, and did not affect the neighboring genes outside it, implicating the existence of region-specific boundaries. Our results suggest that DNA methylation plays important roles in both long-range gene silencing and lineage-specific silencing in embryogenesis.
Dephosphorylation of phosphotyrosine on STAT1 dimers requires extensive spatial reorientation of the monomers facilitated by the N-terminal domain
Authors: Mertens Claudia , Zhong Minghao , Krishnaraj Ravi , Zou Wenxin , Chen Xiaomin , Darnell James E.
Published: December 2006

We report experiments that infer a radical reorientation of tyrosine-phosphorylated parallel STAT1 dimers to an antiparallel form. Such a change in structure allows easy access to a phosphatase. With differentially epitope-tagged molecules, we show that the two monomers of a dimer remain together during dephosphorylation although they most likely undergo spatial reorientation. Extensive single amino acid mutagenesis within crystallographically established domains, manipulation of amino acids in an unstructured tether that connects the N-terminal domain (ND) to the core of the protein, and the demonstration that overexpressed ND can facilitate dephosphorylation of a core molecule lacking an ND all support this model: When the tyrosine-phosphorylated STAT1 disengages from DNA, the ND dimerizes and somehow assists in freeing the reciprocal pY–SH2 binding between the monomers of the dimer while ND ⋅ ND dimerization persists. The core of the monomers rotate allowing reciprocal association of the coiled:coil and DNA-binding domains to present pY at the two ends of an antiparallel dimer for ready dephosphorylation.
FAIRE (Formaldehyde-Assisted Isolation of Regulatory Elements) isolates active regulatory elements from human chromatin
Authors: Giresi Paul G. , Kim Jonghwan , McDaniell Ryan M. , Iyer Vishwanath R. , Lieb Jason D.
Published: December 2006

DNA segments that actively regulate transcription in vivo are typically characterized by eviction of nucleosomes from chromatin and are experimentally identified by their hypersensitivity to nucleases. Here we demonstrate a simple procedure for the isolation of nucleosome-depleted DNA from human chromatin, termed FAIRE (Formaldehyde-AssistedIsolation ofRegulatoryElements). To perform FAIRE, chromatin is crosslinked with formaldehyde in vivo, sheared by sonication, and phenol-chloroform extracted. The DNA recovered in the aqueous phase is fluorescently labeled and hybridized to a DNA microarray. FAIRE performed in human cells strongly enriches DNA coincident with the location of DNaseI hypersensitive sites, transcriptional start sites, and active promoters. Evidence for cell-type–specific patterns of FAIRE enrichment is also presented. FAIRE has utility as a positive selection for genomic regions associated with regulatory activity, including regions traditionally detected by nuclease hypersensitivity assays.
Auditor–client Interaction and Client Usefulness – A Swedish Case Study
Authors: Hellman Niclas
Published: December 2006

This paper investigates the usefulness of the external audit to a listed client company. The research questions focus on what the auditors discovered, the subsequent auditor–client interaction, and the ways in which this was useful to the client company. The study proceeds from the management letters produced by the auditors in the period 1999–2001. On the basis of these reports, interviews were conducted with financial managers at different organisational levels. The data collection and analysis of the data follows a grounded theory approach. The results suggest that the usefulness of the audit to the client company was primarily linked to the way the management letters, and the auditor–client interaction related to the management letters, supported the client’s management control system. Improved management control was achieved as a result of co‐operation between the audit firm and the client company’s central accounting and finance department, that put pressure on the subordinate units. The reported benefits for the client company must be weighed against potential threats to auditor independence, and the paper also includes empirical results that indicate risks of auditor dependency on the client’s accounting and finance department.
Effect of standard medication on quality of life of patients with atopic dermatitis
Authors: KAWASHIMA Makoto , HARADA Shotaro
Published: December 2006

Patients with atopic dermatitis present with debilitating symptoms, including pruritus and subsequent excoriation, which significantly reduces their quality of life (QOL). At present, the standard therapy for atopic dermatitis constitutes a topical steroid and/or a topical immunomodulator, an emollient and an oral antihistamine, although few studies have reported the effect of this treatment regimen on QOL. The current study aimed to verify the efficacy of the standard therapy for both clinical symptom severity and patient QOL, assessed using the validated Skindex‐16 questionnaire. Atopic dermatitis patients receiving the standard therapy (n = 771) were enrolled in the current phase IV, multicenter, 12‐week, open‐label study. The Rajka and Langeland scale (used to rate the severity of atopic dermatitis symptoms) and the Skindex‐16 QOL questionnaire were completed at weeks 0 (baseline), 4 and 12. Of 415 patients completing the questionnaire at all time points (per‐protocol population), 95.2% were prescribed the antihistamine fexofenadine HCl 60 mg. There were significant improvements in symptoms, emotions and functioning scale scores at weeks 4 and 12 compared with baseline (P < 0.005). Discomfort associated with itching, as assessed by item 1 on the Skindex‐16, improved over the treatment period (score decreased by 1 and 2 in 75.2% and 50.9% of patients, respectively). Significant (P < 0.005) improvements from baseline in global scores were also observed at weeks 4 and 12, and for week 12 compared with week 4. Severity scores improved significantly (P < 0.005) from weeks 0–4 and from weeks 4–12. The standard therapy was generally well tolerated with only mild adverse events reported (0.5%). These data suggest that patients with atopic dermatitis and associated pruritus experience significant improvements in both symptom severity and QOL when receiving standard therapy.
Effectiveness of modified phenol peel (Exoderm) on facial wrinkles, acne scars and other skin problems of Asian patients
Authors: PARK Jeong‐Hoon , CHOI Yun‐Duck , KIM Sung‐Wan , KIM Yang‐Che , PARK Sung‐Wook
Published: December 2006

Deep depth peeling using phenol is one of the most effective chemical peeling methods. However, it has been rarely used on Asian people because of side‐effects, especially hypopigmentation. In order to reduce side‐effects, a number of modified phenol peeling formulas were developed. Exoderm, a modified phenol peel, has shown reduced side‐effects such as arrhythmia, long‐lasting erythema, hypopigmentation, hypertrophic scar and keloid. In the present study, our aim was to evaluate the effectiveness of Exoderm on wrinkles, acne scars, and melasma and its safety for Asian patients. Forty‐six patients were treated with Exoderm. Twenty‐eight patients (61%) were treated for wrinkles and 11 patients (24%) for acne scars. Clinical improvement and side‐effects were evaluated and the degree of improvement was graded into four stages. The overall average improvement scores were 3.39 and 3.30 out of 4.00 as evaluated by the doctor and patient, respectively. Forty‐one patients (89%) showed 51% or more improvement. All patients with wrinkles improved 51% or more and their average improvement score was 3.64 out of 4.00. Seven of 11 patients (64%) with acne scars improved 51% or more and their average improvement score was 2.73 out of 4.00. The most frequent side‐effect was postinflammatory hyperpigmentation (74%). Prolonged erythema, keloid and milia were observed temporarily, however, hypopigmentation persisted for 6 months in the follow‐up period. All side‐effects were tolerable and reversible except for one complication of hypopigmentation. We conclude that Exoderm is very effective for treating the facial wrinkles and acne scars of Asian patients.