bluuebunny/crossref_metadata_2025 huggingface dataset:
Antibody C219 recognizes an α-helical epitope on P-glycoprotein
Authors: van den Elsen Jean M. H. , Kuntz Douglas A. , Hoedemaeker Flip J. , Rose David R.
Published: July 2002

The ABC transporter, P-glycoprotein, is an integral membrane protein that mediates the ATP-driven efflux of drugs from multidrug-resistant cancer and HIV-infected cells. Anti-P-glycoprotein antibody C219 binds to both of the ATP-binding regions of P-glycoprotein and has been shown to inhibit its ATPase activity and drug binding capacity. C219 has been widely used in a clinical setting as a tumor marker, but recent observations of cross-reactivity with other proteins, including the c-erbB2 protein in breast cancer cells, impose potential limitations in detecting P-glycoprotein. We have determined the crystal structure at a resolution of 2.4 Å of the variable fragment of C219 in complex with an epitope peptide derived from the nucleotide binding domain of P-glycoprotein. The 14-residue peptide adopts an amphipathic α-helical conformation, a secondary structure not previously observed in structures of antibody–peptide complexes. Together with available biochemical data, the crystal structure of the C219-peptide complex indicates the molecular basis of the cross-reactivity of C219 with non-multidrug resistance-associated proteins. Alignment of the C219 epitope with the recent crystal structure of the ATP-binding subunit of histidine permease suggests a structural basis for the inhibition of the ATP and drug binding capacity of P-glycoprotein by C219. The results provide a rationale for the development of C219 mutants with improved specificity and affinity that could be useful in antibody-based P-glycoprotein detection and therapy in multidrug resistant cancers.
Sorting by diffusion: An asymmetric obstacle course for continuous molecular separation
Authors: Chou Chia-Fu , Bakajin Olgica , Turner Stephen W. P. , Duke Thomas A. J. , Chan Shirley S. , Cox Edward C. , Craighead Harold G. , Austin Robert H.
Published: July 2002

A separation technique employing a microfabricated sieve has been demonstrated by observing the motion of DNA molecules of different size. The sieve consists of a two-dimensional lattice of obstacles whose asymmetric disposition rectifies the Brownian motion of molecules driven through the device, causing them to follow paths that depend on their diffusion coefficient. A nominal 6% resolution by length of DNA molecules in the size range 15–30 kbp may be achieved in a 4-inch (10-cm) silicon wafer. The advantage of this method is that samples can be loaded and sorted continuously, in contrast to the batch mode commonly used in gel electrophoresis.
AF5q31
, a newly identified
AF4
-related gene, is fused to
MLL
in infant acute lymphoblastic leukemia with ins(5;11)(q31;q13q23)
Authors: Taki Tomohiko , Kano Hirotsugu , Taniwaki Masafumi , Sako Masahiro , Yanagisawa Masayoshi , Hayashi Yasuhide
Published: July 2002

Infant acute lymphoblastic leukemia (ALL) with
MLL
gene rearrangements is characterized by early pre-B phenotype (CD10
−
/CD19
+
) and poor treatment outcome. The t(4;11), creating
MLL-AF4
chimeric transcripts, is the predominant 11q23 chromosome translocation in infant ALL and is associated with extremely poor prognosis as compared with other 11q23 translocations. We analyzed an infant early preB ALL with ins(5;11)(q31;q13q23) and identified the
AF5q31
gene on chromosome 5q31 as a fusion partner of the
MLL
gene. The
AF5q31
gene, which encoded a protein of 1,163 aa, was located in the vicinity of the cytokine cluster region of chromosome 5q31 and contained at least 16 exons. The
AF5q31
gene was expressed in fetal heart, lung, and brain at relatively high levels and fetal liver at a low level, but the expression in these tissues decreased in adults. The AF5q31 protein was homologous to AF4-related proteins, including AF4, LAF4, and FMR2. The AF5q31 and AF4 proteins had three homologous regions, including the transactivation domain of AF4, and the breakpoint of AF5q31 was located within the region homologous to the transactivation domain of AF4. Furthermore, the clinical features of this patient with the
MLL-AF5q31
fusion transcript, characterized by the early pre-B phenotype (CD10
−
/CD19
+
) and poor outcome, were similar to those of patients having
MLL-AF4
chimeric transcripts. These findings suggest that AF5q31 and AF4 might define a new family particularly involved in the pathogenesis of 11q23-associated-ALL.
Growth hormone-releasing hormone: An autocrine growth factor for small cell lung carcinoma
Authors: Kiaris H. , Schally A. V. , Varga J. L. , Groot K. , Armatis P.
Published: July 2002

Antagonists of growth hormone-releasing hormone (GHRH) inhibit the growth of various cancers
in vivo
. This effect is thought to be exerted through suppression of the pituitary growth hormone–hepatic insulin-like growth factor I (IGF-I) axis and direct inhibition of autocrine/paracrine production of IGF-I and -II in tumors. However, other evidence points to a direct effect of GHRH antagonists on tumor growth that may not implicate IGFs, although an involvement of GHRH in the proliferation of cancer cells has not yet been established. In the present study we investigated whether GHRH can function as an autocrine/paracrine growth factor in small cell lung carcinoma (SCLC). H-69 and H-510A SCLC lines cultured
in vitro
express mRNA for GHRH, which apparently is translated into peptide GHRH and then secreted by the cells, as shown by the detection of GHRH-like immunoreactivity in conditioned media from the cells cultured
in vitro
. In addition, the levels of GHRH-like immunoreactivity in serum from nude mice bearing H-69 xenografts were higher than in tumor-free mice. GHRH(1–29)NH
2
stimulated the proliferation of H-69 and H-510A SCLCs
in vitro,
and GHRH antagonist JV-1–36 inhibited it. JV-1–36 administered s.c. into nude mice bearing xenografts of H-69 SCLC reduced significantly (
P
< 0.05) tumor volume and weight, after 31 days of therapy, as compared with controls. Collectively, our results suggest that GHRH can function as an autocrine growth factor in SCLCs. Treatment with antagonistic analogs of GHRH may offer a new approach to the treatment of SCLC and other cancers.
Dissecting Fas signaling with an altered-specificity death-domain mutant: Requirement of FADD binding for apoptosis but not Jun N-terminal kinase activation
Authors: Chang Howard Y. , Yang Xiaolu , Baltimore David
Published: July 2002

Fas is a cell surface death receptor that regulates peripheral tolerance and lymphoid homeostasis. In many pathologic conditions, ectopic Fas activation mediates tissue destruction. Several proteins that can bind to the cytoplasmic death domain of Fas have been implicated in Fas signal transduction. Here we show that FADD, which couples Fas to pro-caspase-8, and, Daxx, which couples Fas to the Jun N-terminal kinase pathway, bind independently to the Fas death domain. We have isolated a death domain mutant, termed FasΔ, that selectively binds Daxx but not FADD. In tranfected tissue culture cells, FasΔ activated Jun N-terminal kinase normally but was impaired in cell death induction. These results suggest that FADD and Daxx activate two independent pathways downstream of Fas and confirm the essential role of FADD binding in apoptosis induction.
Protein footprinting at cysteines: Probing ATP-modulated contacts in cysteine-substitution mutants of yeast DNA topoisomerase II
Authors: Tu Benjamin P. , Wang James C.
Published: July 2002

Cysteine-substitution mutants of yeast DNA topoisomerase II were used to test footprinting of the enzyme by 2-nitro-5-thiocyanobenzoate, which cyanylates exposed cysteines in a native protein for peptide cleavage at the cyanylated sites upon unfolding and incubating the protein at pH 9. For a mutant enzyme containing a single cysteine, the extent of peptide cleavage was found to reflect the accessibility of the residue in the native protein. For proteins with multiple cysteines, however, such a correlation was obscured by the transfer of cyano groups from modified to unmodified cysteines during incubation of the unfolded protein at pH 9; accessibilities of the cysteinyl residues in a native protein could be assessed only if cyano shuffling was prevented by blocking uncyanylated sulfhydryls with a second thiol reagent. The successive use of two reagents in cysteine footprinting was applied in probing the ATP-modulated formation of contacts in yeast DNA topoisomerase II.
In vitro
correlates of HIV-2-mediated HIV-1 protection
Authors: Kokkotou Efi G. , Sankalé Jean-Louis , Mani Indu , Guèye-Ndiaye Aissatou , Schwartz David , Essex Myron E. , Mboup Souleymane , Kanki Phyllis J.
Published: July 2002

A prospective study of high-risk commercial sex workers in Senegal has shown that HIV-2 infection may reduce the risk of subsequent HIV-1 infection; these findings have been confirmed and extended, now with 13 years of observation. While exploring the biological mechanisms behind this natural protection, we found that a significant proportion of peripheral blood mononuclear cells obtained from HIV-2-infected subjects resisted
in vitro
challenge with CCR5-dependent HIV-1 viruses but not CXCR4-dependent viruses. High levels of β-chemokines, the natural ligands of the CCR5 coreceptor, were correlated with low levels of viral replication, and resistance was abrogated by antibodies to β-chemokines. Our results suggest that β-chemokine-mediated resistance may be an important correlate of HIV protection against HIV-1 infection and relevant to HIV vaccine design.
Identification of the vitamin K-dependent carboxylase active site: Cys-99 and Cys-450 are required for both epoxidation and carboxylation
Authors: Pudota B. Nirmala , Miyagi Masaru , Hallgren Kevin W. , West Karen A. , Crabb John W. , Misono Kunio S. , Berkner Kathleen L.
Published: July 2002

The vitamin K-dependent carboxylase modifies and renders active
vitamin K-dependent proteins involved in hemostasis, cell growth
control, and calcium homeostasis. Using a novel mechanism, the
carboxylase transduces the free energy of vitamin K hydroquinone
(KH
2
) oxygenation to convert glutamate into a carbanion
intermediate, which subsequently attacks CO
2
, generating
the γ-carboxylated glutamate product. How the carboxylase effects
this conversion is poorly understood because the active site has not
been identified. Dowd and colleagues [Dowd, P., Hershline, R., Ham,
S. W. & Naganathan, S. (1995)
Science
269,
1684–1691] have proposed that a weak base (cysteine) produces a
strong base (oxygenated KH
2
) capable of generating the
carbanion. To define the active site and test this model, we identified
the amino acids that participate in these reactions.
N-
ethyl maleimide inhibited epoxidation and carboxylation,
and both activities were equally protected by KH
2
preincubation. Amino acid analysis of
14
C-
N-
ethyl maleimide-modified human carboxylase revealed
1.8–2.3 reactive residues and a specific activity of 7 ×
10
8
cpm/hr per mg. Tryptic digestion and liquid
chromatography electrospray mass spectrometry identified Cys-99 and
Cys-450 as active site residues. Mutation to serine reduced both
epoxidation and carboxylation, to 0.2% (Cys-99) or 1% (Cys-450), and
increased the
K
m
s for a glutamyl substrate 6-
to 8-fold. Retention of some activity indicates a mechanism for
enhancing cysteine/serine nucleophilicity, a property shared by many
active site thiol enzymes. These studies, which represent a
breakthrough in defining the carboxylase active site, suggest a revised
model in which the glutamyl substrate indirectly coordinates at least
one thiol, forming a catalytic complex that ionizes a thiol to initiate
KH
2
oxygenation.
Prostacyclin-mediated activation of peroxisome proliferator-activated receptor δ in colorectal cancer
Authors: Gupta Rajnish A. , Tan Jian , Krause Wade F. , Geraci Mark W. , Willson Timothy M. , Dey Sudhansu K. , DuBois Raymond N.
Published: July 2002

There is evidence from both genetic and pharmacologic studies
to suggest that the cyclooxygenase-2 (COX-2) enzyme plays a causal role
in the development of colorectal cancer. However, little is known about
the identity or role of the eicosanoid receptor pathways activated by
COX-derived prostaglandins (PG). We previously have reported that
COX-2-derived prostacyclin promotes embryo implantation in the mouse
uterus via activation of the nuclear hormone receptor peroxisome
proliferator-activated receptor (PPAR) δ. In light of the recent
finding that PPARδ is a target of β-catenin transactivation, it is
important to determine whether this signaling pathway is operative
during the development of colorectal cancer. Analysis of PPARδ mRNA
in matched normal and tumor samples revealed that expression of
PPARδ, similar to COX-2, is up-regulated in colorectal carcinomas.
In situ
hybridization studies demonstrate that PPARδ
is expressed in normal colon and localized to the epithelial cells at
the very tips of the mucosal glands. In contrast, expression of PPARδ
mRNA in colorectal tumors was more widespread with increased levels in
transformed epithelial cells. Analysis of PPARδ and COX-2 mRNA in
serial sections suggested they were colocalized to the same region
within a tumor. Finally, transient transfection assays established that
endogenously synthesized prostacyclin (PGI
2
)
could serve as a ligand for PPARδ. In addition, the stable
PGI
2
analog, carbaprostacyclin, and a synthetic PPARδ
agonist induced transactivation of endogenous PPARδ in
human colon carcinoma cells. We conclude from these observations that
PPARδ, similar to COX-2, is aberrantly expressed in colorectal tumors
and that endogenous PPARδ is transcriptionally responsive
to PGI
2
. However, the functional consequence of PPARδ
activation in colon carcinogenesis still needs to be determined.
Pension prospects of minority ethnic groups: inequalities by gender and ethnicity
Authors: Ginn Jay , Arber Sara
Published: July 2002

Minority ethnic groups have low income in later life from private pensions, partly due to shorter employment records in Britain since migration. Yet disadvantage and discrimination in the labour market, as well as differences in cultural norms concerning women's employment, may lead to persistence of ethnic variation in private pension acquisition. Little is known about the pension arrangements made by men and women in minority ethnic groups during the working life.

This paper examines the extent of ethnic disadvantage in private pension scheme arrangements and analyses variation according to gender and specific ethnic group, using three years of the British Family Resources Survey, which provides information on over 97,000 adults aged 20–59, including over 5,700 from ethnic minorities.

Both men and women in minority ethnic groups were less likely to have private pension coverage than their white counterparts but the extent of the difference was most marked for Pakistanis and Bangladeshis. Ethnicity interacted with gender, so that Blacks showed the least gender inequality in private pension arrangements, reflecting the relatively similar full‐time employment rates of Black men and women. A minority ethnic disadvantage in private pension coverage, for both men and women, remained after taking account of age, marital and parental status, years of education, employment variables, class and income.

The research suggests that minority ethnic groups – especially women – will be disproportionately dependent on means‐tested benefits in later life, due to the combined effects of low private pension coverage and the policy of shifting pension provision towards the private sector.
Forum: New Directions in Belarusian Studies Besieged Past: National and Court Historians in Lukashenka's Belarus
Authors: Lindner Rainer
Published: July 2002

At the beginning of the 1990s, when the former Soviet republics declared sovereignty, the questions of their national histories, long neglected in the Soviet period, once again became important. In taking up the national and cultural traditions of the pre-Soviet era, as well as a literary language that had been reduced to folklore, the post-Soviet national intelligentsias began to develop their own versions of the Belarusian past. As the old Soviet empire declined, new “historical” nations developed against a background of diverse ethnicity and political struggles for power. Western scholars have discussed in detail the changes in historical writing since the emergence ofglasnost'. The post-Soviet intelligentsia not only faced a crisis in historical writing and history generally within the late Soviet Union, but were confronted with what Aaron Gurevich has called a “vacuum of historical vision.”
Sin beyond Surfers? Young people's risky behaviour during Schoolies Week in three Queensland regions
Authors: Zinkiewicz L. , Davey J. , Curd D.
Published: July 2002

The present study investigated substance use and interpersonal conflict within the previous 24 hours of young people celebrating Schoolies Week in the Queensland Gold Coast, Sunshine Coast or Wide Bay regions. Questionnaires were completed by 291 males and 367 females, who were predominantly 17 or 18 years old and Queensland residents. Participants engaged in very high levels of drinking, marijuana and ecstasy use in comparison to relevant population statistics. Males were heavier marijuana users and drinkers than were females, and suffered more negative consequences of drinking. They also experienced more interpersonal conflict, although females were more likely to report being sexually harassed. Behaviour was riskier at the Gold Coast than elsewhere, with males within regions engaging in riskier behaviour. Drinkers were more likely to report using marijuana and alcohol, and suffering from the consequences of alcohol, than were non‐drinkers. The data suggest that harm minimization interventions targeted at Schoolies Week participants are needed in all areas in which Schoolies Week are celebrated, although there is a particular need at the Gold Coast. These data also suggest that current interventions may be inadequate in preventing harm to participants, and may need to focus on participants' expectations of the consequences of celebrations.
Psychiatric Disorders and Impairment in the Children of Opiate Addicts: Prevalances and Distribution by Ethnicity
Authors: Nunes Edward V. , Weissman Myrna M. , Goldstein Rise , McAvay Gail , Beckford Carla , Seracini Angela , Verdeli Helena , Wickramaratne Priya
Published: July 2002

This study examined rates of psychiatric disorders and impairment in 283 children, aged 6 to 17, of 69 Caucasian, 45 African‐American, and 47 Hispanic‐American methadone maintenance patients. Children were evaluated by direct and/or parental interview with the K‐SA DS‐E. Final DSM‐III‐R diagnoses and Global Assessment Scale (C‐GAS) were assigned by best estimate. Substantial lifetime prevalences of mood (21%), anxiety (24%) and disruptive disorders (30%), school problems (37%), and global impairment (C‐GAS < 61) (25%) were observed in the children of opiate‐dependent patients. There were few differences between ethnic groups. Effects of proband gender and major depression and their interactions with ethnicity on risk for childhood psychopathology were also examined. The results suggest children of patients in treatment for opiate dependence from diverse ethnic groups are at risk for psychopathology. Programs for early detection and intervention should be devised and evaluated.
Liver Diseases by Alcohol and Hepatitis C: Early Detection and New Insights in Pathogenesis Lead to Improved Treatment
Authors: Lieber Charles S.
Published: July 2002

Much progress has been made in the understanding of the pathogenesis of alcoholic liver disease, resulting in improvement of treatment. Therapy must include correction of nutritional deficiencies, while taking into account changes of nutritional requirements. Methionine is normally activated to S‐adenosylmethionine (SAMe). However, in liver disease, the corresponding enzyme is depressed. The resulting deficiencies can be attenuated by the administration of SAMe but not by methionine. Similarly, phosphatidylethanolamine methyltransferase activity is depressed, but the lacking phosphatidylcholine (PC) can be administrated as polyenylphosphatidylcholine (PPC). Chronic ethanol consumption increases CYP2E1, resulting in increased generation of toxic acetaldehyde and free radicals, tolerance to ethanol and other drugs, and multiple ethanol‐drug interactions. Experimentally, PPC opposes CYP2E1 induction and fibrosis. Alcoholism and hepatitis C infection commonly co‐exist, with acceleration of fibrosis, cirrhosis, and hepatocellular carcinoma. PPC is being tested clinically as a corresponding antifibrotic agent. Available antiviral agents are contraindicated in the alcoholic. Anti‐inflammatory agents, such as steroids, may be selectively useful. Finally, anticraving agents, such as naltrexone or acamprosate, should be part of therapy.
Predictors of Change in Frequency of Crack Cocaine Use in a Street‐Recruited Sample
Authors: Deren Sherry , Beardsley Mark , Tortu Stephanie , Goldstein Marjorie F.
Published: July 2002

Crack cocaine users are at high risk for HIV, with higher frequency crack users engaging in higher rates of HIV‐related sexual risk behaviors. This study will assess the variables impacting changes in crack use frequency. Out‐of‐treatment crack users were street recruited in East Harlem, NY. Subjects (n = 727) were 33% female, 91% minority, and 28% reported recent drug injecting. Baseline and 6‐month follow‐up interviews were administered. There was a significant reduction in crack use over time (p < .0001). Subjects were categorized according to five groups, based on their change in level of crack use between the two interviews, to predict those who stopped, maintained, or changed their level of use. Discriminant analyses identified six variables as the best predictors of the five groups, including having been in drug treatment since baseline and having been a drug injector (both related to reduced levels of crack use). The overall reduction in crack use for the sample masked the fact that important subgroups remained at high use levels or increased their use. The identification of subgroups who may be most resistant to reducing drug use can be helpful in developing more effective interventions.
Subchronic and Chronic Toxicity of Tazarotene Gel Following Topical Application in Hanford Miniswine
Authors: Ismail I. Maged , Baker Brian , Matsumoto Richard , Saaty Tami , Sun Helen , Tang-Liu Diane , Brar Bal
Published: July 2002

Tazarotene, a member of a novel class of synthetic acetylenic retinoids, is the first topical retinoid to demonstrate both efficacy and an acceptable patient tolerability profile in the treatment of psoriasis. The subchronic and chronic toxicity of topically applied tazarotene gel (0.025-0.1%; 0.05-0.5 mg/kg/d) was evaluated in Hanford miniswine in two separate, prospective, randomized, vehicle-controlled studies of 3 months' and 12 months' duration. Although tazarotenic acid, the active metabolite of tazarotene, was detected in the blood of animals in both studies, no signs of systemic toxicity could be detected in hematology, blood chemistry, posttreatment physical examination, necropsy, or histopathological examination of organ tissues. In both studies, the reversible signs and symptoms of cutaneous irritation caused by topical application of tazarotene gel were consistent with those effects commonly produced by other topical and oral retinoids.
Effects of Aldose Reductase Inhibitors, Al-4114 and Al-1576, on Hepatic Biotransformation in Rabbit and Rat
Authors: Veltman J. C. , Kumar A. , Sanders R. A. , Watkins J. B.
Published: July 2002

Aldose reductase is part of the sorbitol pathway, which has been linked to many diabetic complications such as retinopathy and cataracts. This study has determined whether two aldose reductase inhibitors, AL-1576 and AL-4114, may be inducersof hepaticbiotransformation in New Zealandwhiterabbits or Sprague-Dawley rats. The drugs were administered either by intraperitoneal injection (ip) once a day for 4 days (10 mg/kg or 50 mg/kg) to rats and rabbits or by topical-ocular dosing three times a day for 14 days to rabbits (0.5% or 5.0%). Rats dosed ip had increased hepatic cytochrome P450 monooxygenase activity toward methoxy count ar in and benzphetamine, glutathione S-transferase activity toward 1-chloro-2,4-dinitrobenzene, and uridine diphosphate (UDP)-glucurono-syltr an sferase activity toward 4-hydroxybiphenyl and 1-naphthol. In rabbits, ip dosing increased only glucuronosyltransferase activity toward 4-hydroxybiphenyl after AL-4114, and no hepatic biotr an sformation enzyme activities were increased after topical-ocular dosing with either AL-4114 or AL-1576. Activities of other enzymes, including P-450 monooxygenase toward benzo(a)pyrene, N-acetyltr an sferase toward 2-aminofluorene and β-naphthylamine, UDP-glucuronosyltransferase toward 4-methylumbelliferone, styreneoxidehydrolase, and 2-naphthol suIfotransferase, were not increased by either topical-ocular or ip dosing with AL-1576 or AL-4114 in either rats or rabbits, although ip dosing with AL-1576 decreased monooxygenase activity toward ethoxyresorufin in rabbit liver. These results indicate that AL-1576 and AL-4114, though inducers of hepatic biotr an sformation in rats, do not induce hepatic biotran sformation in rabbits when administered by either ip or topical-ocular dosing.
MPTP-Induced Modulation of Neurotransmitters in SH-SY5Y Human Neuroblastoma Cells
Authors: Song Xiaoou , Ehrich Marion
Published: July 2002

Neurotoxic effects of MPTP(1-methyl-4-phenyl-1, 2, 3, 6-tetrahydropyridine) were evaluated in vitro using a human neuronal cell line, SH-SY5Y, that contained features contributing to expression of MPTP toxicity in vivo, namely, a transport system for dopam ine (DA) and monam ine oxidase (MAO) activity. In this model system, MPTP was found to reduce levels of catecholamines (DA, norepinephrine, epinephrine), serotonin (5-HT), and the 5-HT metabolite 5-hydroxyindoleacetic acid (5-HIAA). MPTP enhanced 3H-DA release, which could contribute to the reduction in DA concentrations seen in these cells. In addition, MPTP inhibited MAO activity (Ki 2.26 X 10-5 M). Pretreatment with the MAO inhibitor pargy-line protected the cells from MPTP-induced alterations of catecholamines and the decrease in 5-HT. In this in vitro model, the cholinergic antagonists atro-pine and A-tubocurarine also protected cells from MPTP-induced alterations of catecholamines. The capability of cholinergic antagonists to prevent the MPTP-induced alterations of catecholamine concentrations suggests a possible cholinergic contribution to MPTP neurotoxicity in this cell line.
Experimental Mathematical Validation of a Novel Concept of Extended 2-Butoxyethanol Autoprotection
Authors: Sawant Sharad D. , Doucet Paul G. , Slob Wout , Blaylock Benny L. , Mehendale Harihara M.
Published: July 2002

Prior administration of a moderately hemolytic dose of 2-butoxyethanol (2-BE) in rats leads to autoprotection against the lethal effects of high doses. Digavalli and Mehendale (Arch. Toxicol. 69:526–532;1995) showed that this autoprotection was due to recovery from the prior episode of hemolysis resulting in a higher proportion of young red blood cells (RBCs), which are more resilient to 2-BE. The objective of this research was to investigate the hypothesis that autoprotection can be entirely explained in terms of such changes in age composition of the RBC population. A simple simulation model was developed to provide predictions of the effect of various 2-BE dosage regimes, which were then experimentally verified. Some model predictions were confirmed by the experiments, but others were distinctly off the mark. The longer sequences (two or three successive episodes) in particular showed unexpected erythropoietic responses. This was further investigated in additional experiments, which also looked at reticulocyte counts and serum levels of erythropoietin (Epo). These showed that the release of reticulocytes following a 2-BE challenge is considerably faster than one would expect from the normal processing time in the bone marrow, and also becomes stronger at each successive challenge, resulting in remarkably high levels of reticulocytosis. It is concluded that changes in age composition of the RBCs cannot fully explain the time course of the hematocrit during consecutive administration of several doses of 2-BE, and that other mechanisms must play an important role as well. The results seem to indicate that after the hematocrit has recovered a buffer of (almost mature) reticulocytes remains available in the bone marrow for several days, which can be released almost immediately when another decrease in hematocrit is evoked.
The effect of tobacco smoking on subjective symptoms of inadequacy (“not holding”) of methadone dose among opiate addicts in methadone maintenance treatment
Authors: Tacke Ulrich , Wolff Kim , Finch Emily , Strang John
Published: July 2002

The objective of this study was to examine the relationship between subjective symptoms of inadequacy of methadone dose (not feeling “held”) and tobacco smoking in patients in methadone maintenance treatment (MMT). This was a cross‐sectional study of smoking behaviour, investigating subjective, physiological and psychological symptoms. The study took place in a community‐based methadone maintenance clinic of a psychiatric teaching hospital in South London. Fifty adult opiate addicts (37 males and 13 females) were on a stable daily methadone dose; the number of cigarettes smoked during the day and previous day of investigation, salivary cotinine measurements and carbon monoxide (CO) from expired air were measured. The Methadone Symptom Checklist (MSC) was used to score withdrawal symptoms encountered in patients not feeling “held” during MMT The Hamilton Anxiety Score was also used. The prevalence of tobacco‐smoking was high (98%), with two‐thirds (68%) smoking self‐fabricated cigarettes (“roll‐ups”). Scores from rating scales measuring symptoms of not being “held” correlated with number of cigarettes smoked the previous day (p < 0.05). A similar correlation was found with the Hamilton Anxiety Score. However, there was no correlation between rating scale scores and either salivary cotinine concentration or CO from expired air. Methadone patients who smoke more are significantly more likely to report problems of not feeling “held” by their methadone dose and they also show a higher level of anxiety. However, this increased cigarette consumption is not reflected in increased salivary continine levels or levels of CO in expired air, and it may be that the raised level of anxiety leads to a smoking‐pattern consisting of frequent lighting‐up of cigarettes or “roll‐ups” which are consumed incompletely and/or not smoked by inhalation.
Effect of bromocriptine on acute ethanol tolerance in UChB rats
Authors: TAMPIER LUTSKE , PRADO CRISTIAN , QUINTANILLA MARIA ELENA , MARDONES JORGE
Published: July 2002

It has been suggested that a higher capacity to develop acute tolerance during a single dose of ethanol may promote higher ethanol consumption in alcohol‐preferring rodents. Several studies have shown that the dopaminergic system may be involved in voluntary ethanol consumption. In the present paper we studied the effect of bromocriptine, a dopaminergic agonist drug, that is known to reduce voluntary consumption of ethanol, on acute tolerance in high (UChB) ethanol consumer rats. Acute tolerance was evaluated in bromocriptine and saline‐treated rats by motor impairment induced by a subnarcotic dose of ethanol of 2.3 g/kg IP using a modified tilting plane test. Results showed a highly significant positive correlation between acute tolerance and the voluntary ethanol consumption by the rat. Bromocriptine treatment decreased ethanol consumption and also decreased acute tolerance development. This adds further support to the postulate that the acquisition of acute tolerance to ethanol may promote increased alcohol consumption. Moreover, these results also suggest that dopaminergic receptors involved in ethanol voluntary consumption may also be in acute tolerance development.
Individual Variation in Hepatic Aldehyde Oxidase Activity
Authors: Al‐Salmy H. S.
Published: July 2002

Aldehyde oxidase (AO) is a molybdo‐flavo enzyme expressed predominantly in the liver, lung, and kidney. AO plays a major role in oxidation of aldehydes, as well as oxidation of various N‐heterocyclic compounds of pharmacological and toxicological importance including antiviral (famciclovir), antimalarial (quinine), antitumour (methotrexate), and nicotine. The aim of this study was to investigate cytosolic aldehyde oxidase activity in human liver.Cytosolic AO was characterised using both the metabolism of N‐[(2‐dimethylamino)ethyl] acridine‐4‐carboxamide (DACA) and benzaldehyde to form DACA‐9(10H)‐acridone (quantified by HPLC with fluorescence detection) and benzoic acid (quantified spectrophotometrically). Thirteen livers (10 female, 3 male) were examined. The intrinsic clearance (Vmax/Km) of DACA varied 18‐fold (0.03‐0.50 ml/min/mg). Vmax ranged from 0.20‐3.10 nmol/ min/mg, and Km ranged from 3.5‐14.2 μM. In the same specimens, the intrinsic clearance for benzaldehyde varied 5‐fold (0.40‐1.8 ml/min/mg). Vmax ranged from 3.60‐12.6 nmol/min/mg and Km ranged from 3.6‐14.6 μM. Furthermore, there were no differences in AO activity between male and female human livers, nor was there any relationship to age of donor (range 29‐73 years), smoking status, or disease status. In conclusion, our results showed that there are variations in AO activity in human liver. These variations in aldehyde oxidase activity might reflect individual variations or they might be due to AO stability during processing and storage.
Microdomain Ca2+ Activation during Exocytosis in Paramecium Cells. Superposition of Local Subplasmalemmal Calcium Store Activation by Local Ca2+ Influx
Authors: Erxleben Christian , Klauke Norbert , Flötenmeyer Matthias , Blanchard Marie-Pierre , Braun Claudia , Plattner Helmut
Published: July 2002

In Paramecium tetraurelia, polyamine-triggered exocytosis is accompanied by the activation of Ca2+-activated currents across the cell membrane (Erxleben, C., and H. Plattner. 1994. J. Cell Biol. 127:935– 945). We now show by voltage clamp and extracellular recordings that the product of current × time (As) closely parallels the number of exocytotic events. We suggest that Ca2+ mobilization from subplasmalemmal storage compartments, covering almost the entire cell surface, is a key event. In fact, after local stimulation, Ca2+ imaging with high time resolution reveals rapid, transient, local signals even when extracellular Ca2+ is quenched to or below resting intracellular Ca2+ concentration ([Ca2+]e ⩽ [Ca2+]i). Under these conditions, quenched-flow/freeze-fracture analysis shows that membrane fusion is only partially inhibited. Increasing [Ca2+]e alone, i.e., without secretagogue, causes rapid, strong cortical increase of [Ca2+]i but no exocytosis. In various cells, the ratio of maximal vs. minimal currents registered during maximal stimulation or single exocytotic events, respectively, correlate nicely with the number of Ca stores available. Since no quantal current steps could be observed, this is again compatible with the combined occurrence of Ca2+ mobilization from stores (providing close to threshold Ca2+ levels) and Ca2+ influx from the medium (which per se does not cause exocytosis). This implies that only the combination of Ca2+ flushes, primarily from internal and secondarily from external sources, can produce a signal triggering rapid, local exocytotic responses, as requested for Paramecium defense.
A Distinct and Parallel Pathway for the Nuclear Import of an mRNA-binding Protein
Authors: Pemberton Lucy F. , Rosenblum Jonathan S. , Blobel Günter
Published: July 2002

Three independent pathways of nuclear import have so far been identified in yeast, each mediated by cognate nuclear transport factors, or karyopherins. Here we have characterized a new pathway to the nucleus, mediated by Mtr10p, a protein first identified in a screen for strains defective in polyadenylated RNA export. Mtr10p is shown to be responsible for the nuclear import of the shuttling mRNA-binding protein Npl3p. A complex of Mtr10p and Npl3p was detected in cytosol, and deletion of Mtr10p was shown to lead to the mislocalization of nuclear Npl3p to the cytoplasm, correlating with a block in import. Mtr10p bound peptide repeat-containing nucleoporins and Ran, suggesting that this import pathway involves a docking step at the nuclear pore complex and is Ran dependent. This pathway of Npl3p import is distinct and does not appear to overlap with another known import pathway for an mRNA-binding protein. Thus, at least two parallel pathways function in the import of mRNA-binding proteins, suggesting the need for the coordination of these pathways.
TGF-β Plays a Key Role in Morphogenesis of the Pancreatic Islets of Langerhans by Controlling the Activity of the Matrix Metalloproteinase MMP-2
Authors: Miralles Francisco , Battelino Tadej , Czernichow Paul , Scharfmann Raphael
Published: July 2002

Islets of Langerhans are microorgans scattered throughout the pancreas, and are responsible for synthesizing and secreting pancreatic hormones. While progress has recently been made concerning cell differentiation of the islets of Langerhans, the mechanism controlling islet morphogenesis is not known. It is thought that these islets are formed by mature cell association, first differentiating in the primitive pancreatic epithelium, then migrating in the extracellular matrix, and finally associating into islets of Langerhans. This mechanism suggests that the extracellular matrix has to be degraded for proper islet morphogenesis. We demonstrated in the present study that during rat pancreatic development, matrix metalloproteinase 2 (MMP-2) is activated in vivo between E17 and E19 when islet morphogenesis occurs. We next demonstrated that when E12.5 pancreatic epithelia develop in vitro, MMP-2 is activated in an in vitro model that recapitulates endocrine pancreas development (Miralles, F., P. Czernichow, and R. Scharfmann. 1998. Development. 125: 1017–1024). On the other hand, islet morphogenesis was impaired when MMP-2 activity was inhibited. We next demonstrated that exogenous TGF-β1 positively controls both islet morphogenesis and MMP-2 activity. Finally, we demonstrated that both islet morphogenesis and MMP-2 activation were abolished in the presence of a pan-specific TGF-β neutralizing antibody. Taken together, these observations demonstrate that in vitro, TGF-β is a key activator of pancreatic MMP-2, and that MMP-2 activity is necessary for islet morphogenesis.
Evidence for a Relatively Random Array of Human Chromosomes on the Mitotic Ring
Authors: Allison David C. , Nestor Andrea L.
Published: July 2002

We used fluorescence in situ hybridization (FISH) to study the positions of human chromosomes on the mitotic rings of cultured human lymphocytes, MRC-5 fibroblasts, and CCD-34Lu fibroblasts. The homologous chromosomes of all three cell types had relatively random positions with respect to each other on the mitotic rings of prometaphase rosettes and anaphase cells. Also, the positions of the X and Y chromosomes, colocalized with the somatic homologues in male cells, were highly variable from one mitotic ring to another. Although random chromosomal positions were found in different pairs of CCD-34Lu and MRC-5 late-anaphases, the separations between the same homologous chromosomes in paired late-anaphase and telophase chromosomal masses were highly correlated. Thus, although some loose spatial associations of chromosomes secondary to interphase positioning may exist on the mitotic rings of some cells, a fixed order of human chromosomes and/or a rigorous separation of homologous chromosomes on the mitotic ring are not necessary for normal mitosis. Furthermore, the relative chromosomal positions on each individual metaphase plate are most likely carried through anaphase into telophase.
Integrin-Associated Protein Stimulates α2β1-Dependent Chemotaxis via GI-Mediated Inhibition of Adenylate Cyclase and Extracellular-Regulated Kinases
Authors: Wang Xue-Qing , Lindberg Frederik P. , Frazier William A.
Published: July 2002

Integrin-associated protein (IAP/CD47) augments the function of α2β1 integrin in smooth muscle cells (SMC), resulting in enhanced chemotaxis toward soluble collagen (Wang, X-Q., and W.A. Frazier. 1998. Mol. Biol. Cell. 9:865). IAP-deficient SMC derived from IAP−/− animals did not migrate in response to 4N1K (KRFYVVMWKK), a peptide agonist of IAP derived from the COOH-terminal domain of thrombospondin-1 (TSP1). When normal SMC were preincubated with 4N1K or an anti-α2β1 function-stimulating antibody, cell migration to soluble collagen was significantly enhanced. 4N1K-induced chemotaxis was blocked by treatment of SMC with pertussis toxin indicating that IAP acts through Gi. In agreement with this, 4N1K evoked a rapid decrease in cAMP levels which was intensified in the presence of collagen, and forskolin and 8-Br-cAMP both inhibited SMC migration stimulated via IAP. 4N1K strongly inhibited extracellular regulated kinase (ERK) activation in SMC attaching to collagen and reduced basal ERK activity in suspended SMC. Pertussis toxin treatment of SMC significantly activated ERK, suggesting that an inhibitory input was alleviated. Inhibition of ERK activity by (a) the MAP kinase kinase (MEK) inhibitor, PD98059, (b) antisense oligonucleotide depletion of ERK, and (c) expression of mitogen-activated protein (MAP) kinase phosphatase-1 in SMC all led to increased migration to collagen, 4N1K, or 4N1K plus collagen. Thus, IAP stimulates α2β1 integrin-mediated SMC migration via Gi-mediated inhibition of ERK activity and suppression of cyclic AMP levels. Both of these signaling pathways could directly modulate the state of the integrin as well as impact downstream components of the cell motility apparatus.
Convergence of αvβ3Integrin–And Macrophage Colony Stimulating Factor–Mediated Signals on Phospholipase Cγ in Prefusion Osteoclasts
Authors: Nakamura Ichiro , Lipfert Lorraine , Rodan Gideon A. , Duong Le T.
Published: July 2002

The macrophage colony stimulating factor (M-CSF) and αvβ3 integrins play critical roles in osteoclast function. This study examines M-CSF– and adhesion-induced signaling in prefusion osteoclasts (pOCs) derived from Src-deficient and wild-type mice. Src-deficient cells attach to but do not spread on vitronectin (Vn)-coated surfaces and, contrary to wild-type cells, their adhesion does not lead to tyrosine phosphorylation of molecules activated by adhesion, including PYK2, p130Cas, paxillin, and PLC-γ. However, in response to M-CSF, Src−/− pOCs spread and migrate on Vn in an αvβ3-dependent manner. Involvement of PLC-γ activation is suggested by using a PLC inhibitor, U73122, which blocks both adhesion- and M-CSF–mediated cell spreading. Furthermore, in Src−/− pOCs M-CSF, together with filamentous actin, causes recruitment of β3 integrin and PLC-γ to adhesion contacts and induces stable association of β3 integrin with PLC-γ, phosphatidylinositol 3-kinase, and PYK2. Moreover, direct interaction of PYK2 and PLC-γ can be induced by either adhesion or M-CSF, suggesting that this interaction may enable the formation of integrin-associated complexes. Furthermore, this study suggests that in pOCs PLC-γ is a common downstream mediator for adhesion and growth factor signals. M-CSF–initiated signaling modulates the αvβ3 integrin-mediated cytoskeletal reorganization in prefusion osteoclasts in the absence of c-Src, possibly via PLC-γ.
A Central Role for the Armadillo Protein Plakoglobin in the Autoimmune Disease Pemphigus Vulgaris
Authors: Caldelari Reto , de Bruin Alain , Baumann Dominique , Suter Maja M. , Bierkamp Christiane , Balmer Vreni , Müller Eliane
Published: July 2002

In pemphigus vulgaris (PV), autoantibody binding to desmoglein (Dsg) 3 induces loss of intercellular adhesion in skin and mucous membranes. Two hypotheses are currently favored to explain the underlying molecular mechanisms: (a) disruption of adhesion through steric hindrance, and (b) interference of desmosomal cadherin-bound antibody with intracellular events, which we speculated to involve plakoglobin. To investigate the second hypothesis we established keratinocyte cultures from plakoglobin knockout (PG−/−) embryos and PG+/+ control mice. Although both cell types exhibited desmosomal cadherin-mediated adhesion during calcium-induced differentiation and bound PV immunoglobin (IgG) at their cell surface, only PG+/+ keratinocytes responded with keratin retraction and loss of adhesion. When full-length plakoglobin was reintroduced into PG−/− cells, responsiveness to PV IgG was restored. Moreover, in these cells like in PG+/+ keratinocytes, PV IgG binding severely affected the linear distribution of plakoglobin at the plasma membrane. Taken together, the establishment of an in vitro model using PG+/+ and PG−/− keratinocytes allowed us (a) to exclude the steric hindrance only hypothesis, and (b) to demonstrate for the first time that plakoglobin plays a central role in PV, a finding that will provide a novel direction for investigations of the molecular mechanisms leading to PV, and on the function of plakoglobin in differentiating keratinocytes.
Eg5 is static in bipolar spindles relative to tubulin
Authors: Kapoor Tarun M. , Mitchison Timothy J.
Published: July 2002

We used fluorescent speckle microscopy to probe the dynamics of the mitotic kinesin Eg5 in Xenopus extract spindles, and compared them to microtubule dynamics. We found significant populations of Eg5 that were static over several seconds while microtubules flux towards spindle poles. Eg5 dynamics are frozen by adenylimidodiphosphate. Bulk turnover experiments showed that Eg5 can exchange between the spindle and the extract with a half life of <55 s. Eg5 distribution in spindles was not perturbed by inhibition of its motor activity with monastrol, but was perturbed by inhibition of dynactin with p50 dynamitin. We interpret these data as revealing the existence of a static spindle matrix that promotes Eg5 targeting to spindles, and transient immobilization of Eg5 within spindles. We discuss alternative interpretations of the Eg5 dynamics we observe, ideas for the biochemical nature of a spindle matrix, and implications for Eg5 function.
Let's make Golgi
Authors: Wells William A.
Published: July 2002

Does the Golgi self-organize or does it form around an instructive template? Evidence on both sides is piling up, but a definitive conclusion is proving elusive.
Role of the Thymus in Transplantation Tolerance in Miniature Swine. I. Requirement of the Thymus for Rapid and Stable Induction of  Tolerance to Class I–mismatched Renal Allografts
Authors: Yamada Kazuhiko , Gianello Pierre R. , Ierino Francesco L. , Lorf Thomas , Shimizu Akira , Meehan Shane , Colvin Robert B. , Sachs David H.
Published: July 2002

The almost uniform failure in transplant patients of tolerance-inducing regimens that have been found to be effective in rodents, has made it necessary to examine large animal models before testing of new approaches clinically. Miniature swine have been shown to share many relevant immunologic parameters with humans, and because of their reproducible genetics, have proved extremely useful in providing such a large animal model. We have previously shown that indefinite systemic tolerance to renal allografts in miniature swine is induced in 100% of cases across a two-haplotype class I plus minor histocompatibility antigen disparity by a 12-d course of Cyclosporine A (CyA), in contrast to irreversible rejection observed uniformly without CyA treatment. In the present study, we have examined the role of the thymus during the induction of tolerance by performing a complete thymectomy 21 d before renal transplantation. This analysis demonstrated a striking difference between thymectomized and nonthymectomized animals. Thymectomized swine developed acute cellular rejection characterized by a T cell (CD25+) infiltrate, tubulitis, endothelialitis and glomerulitis, and anti–donor CTL reactivity in vitro. Nonthymectomized and sham thymectomized animals had a mild T cell infiltrate with few CD25+ cells and no anti–donor CTL response in vitro. These results indicate that the thymus is required for rapid and stable induction of tolerance.