bluuebunny/crossref_metadata_2025 huggingface dataset:
Vulnerability of family caregivers in terminal palliative care at home; balancing between burden and capacity
Authors: Proot Ireen M. , Abu‐Saad Huda Huijer , Crebolder Harry F.J.M. , Goldsteen Minke , Luker Karen A. , Widdershoven Guy A.M.
Published: May 2003

This paper reports on a grounded theory interview‐based study with 13 family members aged 28–80 years caring for terminally ill people at home (with a life expectancy of 3 months or less) in the Netherlands. The project was approved by the ethics committee of the Maastricht University Hospital. The aim of this study was to explore the experiences of family caregivers, their needs for home care, and which health services they receive. Data were analysed using the constant comparative method. ‘Vulnerability’ was identified as the core category. Caring for a terminally ill person at home requires continuous balancing between care burden and capacity to cope. Whether or not the carer will succeed in keeping in optimum balance is dependent on a number of factors impinging on the caregiver's vulnerability. Care burden, restricted activities, fear, insecurity, loneliness, facing death, lack of emotional, practical and information‐related support were identified from the data as factors having the potential to increase the caregiver's vulnerability, and may be risk factors for fatigue and burnout. Continuing previous activities, hope, keeping control, satisfaction and good support are factors which may decrease the caregiver's vulnerability, and may protect against fatigue and burnout. The experiences of the caregivers in our study showed that the support from informal and professional caregivers was not sufficient. Education and practical tools may make professionals more sensitive for the vulnerable position of family caregivers, even when these caregivers do not show their vulnerability.
Sequence and structural analysis of the presumed downstream promoter of the canine mdr1 gene
Authors: Mealey K. L. , Bentjen S. A.
Published: May 2003

The product of the canine mdr1 gene, P‐glycoprotein (P‐gp), plays an important role in chemotherapeutic drug resistance of several canine tumours. Increased expression of P‐gp by tumour cells is associated with the multidrug‐resistant phenotype. Because of its importance in cancer chemotherapy, a great deal is known about the regulation of mdr1 gene expression in human cancer patients and rodent cancer models. In contrast, there is no information regarding the regulation of P‐gp expression in dogs. Initial information regarding the regulation of mdr1 gene expression can be gained by evaluating the mdr1 promoter. The downstream promoter of the canine mdr1 gene was sequenced. Several regulatory elements were identified, including an AP‐1 site, AP‐2 site and SP‐1 site. The presumed canine mdr1 promoter was similar to that of other species; however, low overall sequence homology may suggest that aspects of P‐gp regulation are distinctive in dogs.
Immunohistochemical characterization of canine prostatic carcinoma and correlation with castration status and castration time
Authors: Sorenmo K. U. , Goldschmidt M. , Shofer F. , Goldkamp C. , Ferracone J.
Published: May 2003

The purpose of this study was to characterize canine prostate cancer using immunohistochemical staining specific for acinar and urothelial/ductal tissue and correlate these results with the dogs' castration status/castration time. Seventy dogs with prostate cancer were included, 71% were castrated and 29% were intact. Compared with an age‐matched control population, castrated dogs were at increased risk of prostate cancer, odds ratio 3.9. Immunohistochemical staining was performed on 58 cases. Forty‐six of the 58 stained positive for cytokeratin 7 (CK 7) (ductal/urothelial origin) and one of the 58 stained positive for prostate‐specific antigen. Dogs with CK 7‐positive tumours were younger when castrated than dogs with CK 7‐negative tumours, 2 versus 7 years (P = 0.03); dogs castrated at ≤2 years of age were more likely to be CK 7‐positive (P = 0.009). These results show that most canine prostatic carcinomas are of ductal/urothelial, androgen‐independent origin. This is consistent with the epidemiological findings, showing increased risk in castrated dogs. Canine prostate cancer may, therefore, not be a realistic model for the human disease.
Mhc Class Ii–Expressing Hepatocytes Function As Antigen–Presenting Cells and Activate Specific Cd4 T Lymphocyutes
Authors: Herkel Johannes , Jagemann Bettina , Wiegard Christiane , Lazaro Jose Francisco Garcia , Lueth Stefan , Kanzler Stephan , Blessing Manfred , Schmitt Edgar , Lohse Ansgar W.
Published: May 2003

The ability to activate CD4 T cells is restricted to antigen–presenting cells that express major histocompatibility complex (MHC) class II molecules. Parenchymal cells normally do not express MHC class II molecules; however, in clinical hepatitis, viral or autoimmune, hepatocytes often exhibit aberrant MHC class II expression. It is not known whether MHC class II–expressing hepatocytes can function as antigen–presenting cells, but it has been suggested that aberrant MHC class II expression by parenchymal cells may cause autoimmune disease. Therefore, we generated transgenic mice that specifically overexpress class II transactivator molecules in hepatocytes. Hepatocytes from these mice exhibited stable MHC class II expression and were used to stimulate CD4 T cells from T–cell receptor transgenic mice and CD4 T–cell lines. MHC II–expressing hepatocytes featured costimulatory CD80 molecules and could serve as antigen–presenting cells that were able to process protein antigen and to activate specific CD4 T cells. Nevertheless, the transgenic mice with aberrant hepatocellular MHC class II expression did not exhibit any symptoms of autoimmune disease. In conclusion, MHC II–expressing hepatocytes, as found in clinical hepatitis, can present antigen and activate CD4 T cells. The ability of hepatocytes to present antigen on MHC II molecules does not seem to be a sufficient cause for inflammatory autoimmunity and hepatitis. However, we still need to explore whether such antigen presentation is occurring
in vivo
. The transgenic mice described in this study may serve as a model to study the immune interaction of hepatocytes and CD4 T cells in both
in vitro
and
in vivo
.
The crystal structure of dipeptidyl peptidase IV (CD26) reveals its functional regulation and enzymatic mechanism
Authors: Engel Michael , Hoffmann Torsten , Wagner Leona , Wermann Michael , Heiser Ulrich , Kiefersauer Reiner , Huber Robert , Bode Wolfram , Demuth Hans-Ulrich , Brandstetter Hans
Published: April 2003

The membrane-bound glycoprotein dipeptidyl peptidase IV (DP IV, CD26) is a unique multifunctional protein, acting as receptor, binding and proteolytic molecule. We have determined the sequence and 1.8 Å crystal structure of native DP IV prepared from porcine kidney. The crystal structure reveals a 2-2-2 symmetric tetrameric assembly which depends on the natively glycosylated β-propeller blade IV. The crystal structure indicates that tetramerization of DP IV is a key mechanism to regulate its interaction with other components. Each subunit comprises two structural domains, the N-terminal eight-bladed β-propeller with open Velcro topology and the C-terminal α/β-hydrolase domain. Analogy with the structurally related POP and tricorn protease suggests that substrates access the buried active site through the β-propeller tunnel while products leave the active site through a separate side exit. A dipeptide mimicking inhibitor complexed to the active site discloses key determinants for substrate recognition, including a Glu–Glu motif that distinguishes DP IV as an aminopeptidase and an oxyanion trap that binds and activates the P2-carbonyl oxygen necessary for efficient postproline cleavage. We discuss active and nonactive site-directed inhibition strategies of this pharmaceutical target protein.
Phage HK022 Nun protein represses translation of phage λ N (transcription termination/translation repression)
Authors: Kim Hyeong C. , Zhou Jian-guang , Wilson Helen R. , Mogilnitskiy Grigoriy , Court Donald L. , Gottesman Max E.
Published: April 2003

The N-terminal arginine-rich motif of phage HK022 Nun protein binds to NUT sequences in phage λ nascent transcripts and induces transcription termination. Interactions between the Nun C terminus and RNA polymerase as well as the DNA template are required for termination. We have isolated Nun C-terminal point and deletion mutants that are unable to block transcription. The mutants bind NUT RNA and inhibit translation of the λ N gene. Thus HK022 excludes λ both by terminating transcription on the phage chromosome and by preventing translation of the essential λ N gene. Like N autoregulation, translation repression by Nun requires host RNaseIII deficiency (
rnc
) or a mutation in the RNaseIII processing site (rIII) located between NUTL and the beginning of the N coding sequence. Our data support the idea that Nun bound at NUTL causes steric interference with ribosome attachment to the nearby N coding sequence. Two models, Nun acting alone or in complex with host proteins, are discussed.
Cloning of wrinkle-free, a previously uncharacterized mouse mutation, reveals crucial roles for fatty acid transport protein 4 in skin and hair development
Authors: Moulson Casey L. , Martin Daniel R. , Lugus Jesse J. , Schaffer Jean E. , Lind Anne C. , Miner Jeffrey H.
Published: April 2003

Wrinkle-free (
wrfr
) is a previously uncharacterized, spontaneous, autosomal recessive mouse mutation resulting in very tight, thick skin.
wrfr
mutant mice exhibit severe breathing difficulties secondary to their tight skin and die shortly after birth. This phenotype is strikingly similar to a very rare human genetic disorder, restrictive dermopathy.
wrfr
mutant mice display a defective skin barrier, which is normally imparted by the cornified envelope, a composite of protein and lipid that prevents loss of water from within and entry of potentially harmful substances from without. In addition, hair growth from grafted
wrfr
skin is impaired. Positional cloning of the
wrfr
mutation revealed a retrotransposon insertion into a coding exon of
Slc27a4
, the gene encoding fatty acid transport protein (FATP)4. FATP4 is the primary intestinal FATP and is thought to play a major role in dietary fatty acid uptake; it therefore is viewed as a target to prevent or reverse obesity. However, its function
in vivo
had not been determined. Our results demonstrate an unexpected yet critical role for FATP4 in skin and hair development and suggest
Slc27a4
to be a candidate gene for restrictive dermopathy.
Cell spreading controls endoplasmic and nuclear calcium: A physical gene regulation pathway from the cell surface to the nucleus
Authors: Itano Naoki , Okamoto Shu-ichi , Zhang Dongxian , Lipton Stuart A. , Ruoslahti Erkki
Published: April 2003

Cells attaching to an extracellular matrix through integrins flatten out (spread) on the matrix, eliciting cellular signals needed for survival. We show that the shape of the nucleus changes and the nuclear calcium level increases in spreading cells. Moreover, cell spreading and osmotic stretching of isolated nuclei cause release of perinuclear Ca
2+
, and patch clamping of nuclei reveals stretch-activated Ca
2+
permeable channels. Gene expression assays with myocyte enhancer factor 2, which is activated by calmodulin-dependent kinase IV, indicate that the elevation in nuclear Ca
2+
is functionally significant. We propose a mechano-transduction pathway in which spreading-induced nuclear stretching releases Ca
2+
from the perinuclear space, and the resulting Ca
2+
elevation in the nucleus provokes changes in gene expression.
The rate of hydrolysis of phosphomonoester dianions and the exceptional catalytic proficiencies of protein and inositol phosphatases
Authors: Lad Chetan , Williams Nicholas H. , Wolfenden Richard
Published: May 2003

To evaluate the proficiency of phosphatases as catalysts, the rate of the uncatalyzed hydrolysis of simple phosphate monoester dianions was estimated by extrapolating rates measured over a range of high temperatures. The rate of spontaneous hydrolysis of phenyl phosphate dianion indicates that a linear free energy relationship reported earlier is reliable for leaving groups whose conjugate acids have pKa values up to at least 10. Using Teflon reaction vessels, it proved possible to follow the hydrolysis of methyl phosphate and 3-(4-carboxy)-2,2-dimethylpropyl phosphate in strong alkali. Even in 1 M KOH, the reaction was found to be specific acid catalyzed. These results establish an upper limit for dianion reactivity, which had been overestimated earlier as a result of the leaching by alkali of silicic acid from quartz reaction vessels. The present findings indicate that the half-time for attack by water on alkyl phosphate dianions is 1.1 × 10
12
years (
k
= 2 × 10
−20
s) at 25°C and that phosphatases involved in cell signaling and regulation produce the largest rate enhancements that have been identified thus far. Protein phosphatase-1 and inositol 1-phosphatase exceed all other known enzymes in their affinities for the altered substrates in the transition state.
The amelogenin loci span an ancient pseudoautosomal boundary in diverse mammalian species
Authors: Iwase Mineyo , Satta Yoko , Hirai Yuriko , Hirai Hirohisa , Imai Hirotami , Takahata Naoyuki
Published: April 2003

The mammalian amelogenin (
AMEL
) genes are found on both the X and Y chromosomes (gametologous). Comparison of the genomic
AMEL
sequences in five primates and three other mammals reveals that the 5′ portion of the gametologous
AMEL
loci began to differentiate in the common ancestor of extant mammals, whereas the 3′ portion differentiated independently within species of different mammals. The boundary is marked by a transposon insertion in intron 2 and is shared by all species examined. In addition, 540-kb DNA sequences from the short arm of the human X chromosome are aligned with their Y gametologous sequences. The pattern and extent of sequence differences in the 5′ portion of the
AMEL
loci extend to a proximal region that contains the
ZFX
locus, and those in the 3′ portion extend all the way down to the pseudoautosomal boundary (PAB)1. We concluded that the
AMEL
locus spans an ancient PAB, and that both the ancient and present PABs were determined by chance events during the evolution of mammals and primates. Sex chromosome differentiation likely took place in a region that contains the male-determining loci by suppressing homologous recombination.
A genetic model for a central (septum transversum) congenital diaphragmatic hernia in mice lacking
Slit3
Authors: Yuan Wenlin , Rao Yi , Babiuk Randal P. , Greer John , Wu Jane Y. , Ornitz David M.
Published: April 2003

Congenital diaphragmatic hernia (CDH) is a significant cause of pediatric mortality in humans with a heterogeneous and poorly understood etiology. Here we show that mice lacking
Slit3
developed a central (septum transversum) CDH.
Slit3
encodes a member of the Slit family of guidance molecules and is expressed predominantly in the mesothelium of the diaphragm during embryonic development. In
Slit3
null mice, the central tendon region of the diaphragm fails to separate from liver tissue because of abnormalities in morphogenesis. The CDH progresses through continuous growth of the liver into the thoracic cavity. This study establishes the first genetic model for CDH and identifies a previously unsuspected role for Slit3 in regulating the development of the diaphragm.
Xylulose 5-phosphate mediates glucose-induced lipogenesis by xylulose 5-phosphate-activated protein phosphatase in rat liver
Authors: Kabashima Tsutomu , Kawaguchi Takumi , Wadzinski Brian E. , Uyeda Kosaku
Published: April 2003

Carbohydrate-responsive element binding protein (ChREBP) is a transcription factor that activates lipogenic genes in liver in response to excess carbohydrate in the diet. ChREBP is regulated in a reciprocal manner by glucose and cAMP. cAMP-dependent protein kinase (protein kinase A) phosphorylates two physiologically important sites in ChREBP, Ser-196, which is located near nuclear localization signal sequence (NLS), and Thr-666, within the basic helix–loop–helix (bHLH) site, resulting in inactivation of nuclear translocation of ChREBP and of the DNA-binding activity, respectively. We demonstrate here that crude cytosolic extracts from livers of rats fed a high carbohydrate diet contained protein phosphatase (PPase) activity that dephosphorylated a peptide containing Ser-196, whereas a PPase in the nuclear extract catalyzed dephosphorylation of Ser-568 and Thr-666. All these PPases are activated specifically by xylulose 5-phosphate (Xu5P), but not by other sugar phosphates. Furthermore, addition of Xu5P elevated PPase activity to the level observed in extracts of fed liver cells. These partially purified PPases were characterized as PP2A-ABδC by immunoblotting with specific antibodies. These results suggest that (
i
a) Xu5P-dependent PPase is responsible for activation of transcription of the L-type pyruvate kinase gene and lipogenic enzyme genes, and (
ii
) Xu5P is the glucose signaling compound. Thus, we propose that the same Xu5P-activated PPase controls both acute and long-term regulation of glucose metabolism and fat synthesis.
Perturbations in B cell responsiveness to CD4+T cell help in HIV-infected individuals
Authors: Moir Susan , Ogwaro Kisani M. , Malaspina Angela , Vasquez Joshua , Donoghue Eileen T. , Hallahan Claire W. , Liu Shuying , Ehler Linda A. , Planta Marie A. , Kottilil Shyamasundaran , Chun Tae-Wook , Fauci Anthony S.
Published: May 2003

HIV infection induces a wide array of B cell dysfunctions. We have characterized the effect of plasma viremia on the responsiveness of B cells to CD4+T cell help in HIV-infected patients. In HIV-negative donors, B cell proliferation correlated with CD154 expression on activated CD4+T cells and with the availability of IL-2, whereas in HIV-infected viremic patients, reduced B cell proliferation was observed despite normal CD154 expression on activated CD4+T cells. Reduced triggering of B cells by activated CD4+T cells was clearly observed in HIV-infected viremic patients compared with aviremic patients with comparable CD4+T cell counts, and a dramatic improvement in B cell function was observed in patients whose plasma viremia was controlled by effective antiretroviral therapy. The degree of B cell dysfunction in viremic patients correlated strongly with the inability of B cells to express CD25 in response to activated CD4+T cells, resulting in an inability to mount a normal proliferative response to IL-2. Similar defects in responsiveness to IL-2 were observed in the B cells of HIV-infected viremic patients in the context of B cell receptor stimulation. These data provide new insight into the mechanisms associated with ineffective humoral responses in HIV disease.
Synthetic matrix metalloproteinase-sensitive hydrogels for the conduction of tissue regeneration: Engineering cell-invasion characteristics
Authors: Lutolf M. P. , Lauer-Fields J. L. , Schmoekel H. G. , Metters A. T. , Weber F. E. , Fields G. B. , Hubbell J. A.
Published: April 2003

Synthetic hydrogels have been molecularly engineered to mimic the invasive characteristics of native provisional extracellular matrices: a combination of integrin-binding sites and substrates for matrix metalloproteinases (MMP) was required to render the networks degradable and invasive by cells via cell-secreted MMPs. Degradation of gels was engineered starting from a characterization of the degradation kinetics (kcatandKm) of synthetic MMP substrates in the soluble form and after crosslinking into a 3D hydrogel network. Primary human fibroblasts were demonstrated to proteolytically invade these networks, a process that depended on MMP substrate activity, adhesion ligand concentration, and network crosslinking density. Gels used to deliver recombinant human bone morphogenetic protein-2 to the site of critical defects in rat cranium were completely infiltrated by cells and remodeled into bony tissue within 4 wk at a dose of 5 μg per defect. Bone regeneration was also shown to depend on the proteolytic sensitivity of the matrices. These hydrogels may be useful in tissue engineering and cell biology as alternatives for naturally occurring extracellular matrix-derived materials such as fibrin or collagen.
Mycobacterium tuberculosisglycosylated phosphatidylinositol causes phagosome maturation arrest
Authors: Fratti Rutilio A. , Chua Jennifer , Vergne Isabelle , Deretic Vojo
Published: April 2003

The tubercle bacillus parasitizes macrophages by inhibiting phagosome maturation into the phagolysosome. This phenomenon underlies the tuberculosis pandemic involving 2 billion people. We report here howMycobacterium tuberculosiscauses phagosome maturation arrest. A glycosylatedM. tuberculosisphosphatidylinositol [mannose-capped lipoarabinomannan (ManLAM)] interfered with the phagosomal acquisition of the lysosomal cargo and syntaxin 6 from the trans-Golgi network. ManLAM specifically inhibited the pathway dependent on phosphatidylinositol 3-kinase activity and phosphatidylinositol 3-phosphate-binding effectors. These findings identify ManLAM as theM. tuberculosisproduct responsible for the inhibition of phagosomal maturation.
γ-Aminobutyric acid B receptor 1 mediates behavior-impairing actions of alcohol in
Drosophila
: Adult RNA interference and pharmacological evidence
Authors: Dzitoyeva Svetlana , Dimitrijevic Nikola , Manev Hari
Published: April 2003

In addition to their physiological function, metabotropic receptors for neurotransmitter γ-aminobutyric acid (GABA), the GABA
B
receptors, may play a role in the behavioral actions of addictive compounds. Recently, GABA
B
receptors were cloned in fruit flies (
Drosophila melanogaster
), indicating that the advantages of this experimental model could be applied to GABA
B
receptor research. RNA interference (RNAi) is an endogenous process triggered by double-stranded RNA and is being used as a tool for functional gene silencing and functional genomics. Here we show how cell-nonautonomous RNAi can be induced in
adult
fruit flies to silence a subtype of GABA
B
receptors, GABA
B
R1, and how RNAi combined with pharmacobehavioral techniques (including intraabdominal injections of active compounds and a computer-assisted quantification of behavior) can be used to functionally characterize these receptors. We observed that injection of double-stranded RNA complementary to GABA
B
R1 into adult
Drosophila
selectively destroys GABA
B
R1 mRNA and attenuates the behavioral actions of the GABA
B
agonist, 3-aminopropyl-(methyl)phosphinic acid. Moreover, both GABA
B
R1 RNAi and the GABA
B
antagonist CGP 54626 reduced the behavior-impairing effects of ethanol, suggesting a putative role for the
Drosophila
GABA
B
receptors in alcohol's mechanism of action. The
Drosophila
model we have developed can be used for further
in vivo
functional characterization of GABA
B
receptor subunits and their involvement in the molecular and systemic actions of addictive substances.
Activation of liver X receptor improves glucose tolerance through coordinate regulation of glucose metabolism in liver and adipose tissue
Authors: Laffitte Bryan A. , Chao Lily C. , Li Jing , Walczak Robert , Hummasti Sarah , Joseph Sean B. , Castrillo Antonio , Wilpitz Damien C. , Mangelsdorf David J. , Collins Jon L. , Saez Enrique , Tontonoz Peter
Published: April 2003

The control of lipid and glucose metabolism is closely linked. The nuclear receptors liver X receptor (LXR)α and LXRβ have been implicated in gene expression linked to lipid homeostasis; however, their role in glucose metabolism is not clear. We demonstrate here that the synthetic LXR agonist GW3965 improves glucose tolerance in a murine model of diet-induced obesity and insulin resistance. Analysis of gene expression in LXR agonist-treated mice reveals coordinate regulation of genes involved in glucose metabolism in liver and adipose tissue. In the liver, activation of LXR led to the suppression of the gluconeogenic program including down-regulation of peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1), phospho
enol
pyruvate carboxykinase (PEPCK), and glucose-6-phosphatase expression. Inhibition of gluconeogenic genes was accompanied by an induction in expression of glucokinase, which promotes hepatic glucose utilization. In adipose tissue, activation of LXR led to the transcriptional induction of the insulin-sensitive glucose transporter, GLUT4. We show that the GLUT4 promoter is a direct transcriptional target for the LXR/retinoid X receptor heterodimer and that the ability of LXR ligands to induce GLUT4 expression is abolished in LXR null cells and animals. Consistent with their effects on GLUT4 expression, LXR agonists promote glucose uptake in 3T3-L1 adipocytes
in vitro
. Thus, activation of LXR alters the expression of genes in liver and adipose tissue that collectively would be expected to limit hepatic glucose output and improve peripheral glucose uptake. These results outline a role for LXRs in the coordination of lipid and glucose metabolism.
N
-methyl-
d
-aspartate receptor blockade during development lowers long-term potentiation threshold without affecting dynamic range of CA3-CA1 synapses
Authors: Savić Nataša , Lüthi Andreas , Gähwiler Beat H. , McKinney R. Anne
Published: April 2003

During development, excitatory synapses in the CA1 region of the hippocampus undergo activity-dependent and
N
-methyl-
d
-aspartate (NMDA) receptor-dependent long-lasting changes in synaptic efficacy. These bidirectional changes occur between limits that determine the dynamic range within which synapses operate. It is unknown whether the dynamic range itself is also activity-dependent and NMDA receptor-dependent. Here, we show that chronic blockade of NMDA receptors in hippocampal slice cultures during early postnatal development does not affect the dynamic range but results in a lower threshold for the induction of long-term potentiation. Thus, the dynamic range of CA3-CA1 synapses, unlike long-term potentiation threshold, is NMDA receptor-independent, thereby providing functional stability to the hippocampal network during development.
An important regulatory role for CD4+CD8αα T cells in the intestinal epithelial layer in the prevention of inflammatory bowel disease
Authors: Das Gobardhan , Augustine Mathew M. , Das Jyoti , Bottomly Kim , Ray Prabir , Ray Anuradha
Published: April 2003

The normal immunoregulatory mechanisms that maintain homeostasis in the intestinal mucosa, despite continuous provocation by environmental antigens, are jeopardized in inflammatory bowel diseases. Although previous studies have suggested that intestinal intraepithelial lymphocytes prevent spontaneous intestinal inflammation, there is limited knowledge about the characteristics of regulatory cells in the intestinal intraepithelial lymphocytes population. Here we show that CD4+CD8αα+double-positive cells present in the intestinal intraepithelial lymphocytes population can suppress T helper 1-induced intestinal inflammation in an IL-10-dependent fashion. CD4+T cells stimulated along the Th2 but not the Th1 lineage, when transferred toRAG-1−/−mice, acquire CD8αα expression on reaching the intestinal epithelium, and on arrival there, augment their production of IL-10. We show that a precursor CD4+T cell after limited, but not repeated, stimulation by IL-4 is able to become a double-positive-regulatory cell on exposure to the intestinal microenvironment in mice. Both CD8αα acquisition and IL-10 production depend critically on the NF-κB-GATA-3-axis that we have previously shown is essential for differentiation to the Th2 phenotype and for the induction of airway inflammation. Our studies identify a mechanism for the generation of regulatory T cells in the intestine that may play an important role in controlling inflammatory bowel disease.
Plasma antioxidant status and cell injury after severe physical exercise
Authors: Chevion Shlomit , Moran Danny S. , Heled Yuval , Shani Yoav , Regev Gilad , Abbou Benny , Berenshtein Eduard , Stadtman Earl R. , Epstein Yoram
Published: April 2003

Strenuous exercise leads to an increase in metabolic rate, increased production of reactive oxygen species, and compromised antioxidant defense systems. To study the effects of oxidative stress during strenuous exercise, a homogeneous group of 31 male subjects participated in a 6-month, 5 days/week training schedule involving two extreme marches of 50 km and 80 km at sea level, separated by 2 weeks of regular training. Each participant carried 35 kg of extra weight. Blood samples were drawn imediately before and after each march. Twenty-nine subjects completed the 50-km march, and only 16 completed the 80-km march. Plasma levels of reduced ascorbic acid, total ascorbate, and dehydroascorbate did not undergo significant changes during either march. However, the 50- and 80-km marches led to 25% and 37% increases, respectively, in plasma levels of uric acid; due presumably to increases in the metabolic rate and consequent pyrimidine nucleotide metabolism. Both marches led to ≈10-fold increase leakage of creatine phosphokinase into the plasma. Likewise, plasma levels of aspartate transaminase, a characteristic marker of liver injury, increased ≈4-fold. Plasma levels of bilirubin, creatine, urea, and glucose also increased. Plasma protein carbonyl content, a marker of protein oxidative damage, decreased significantly during each march. These results are discussed with respect to the consideration that elevation of the respiration rate during exercise leads to production of more reactive oxygen species than the antioxidant systems can scavenge. Plausible explanations for leakage of molecules into the plasma are discussed.
Oligonucleotide microarrays demonstrate the highest frequency of
ATM
mutations in the mantle cell subtype of lymphoma
Authors: Fang Nicole Y. , Greiner Timothy C. , Weisenburger Dennis D. , Chan Wing C. , Vose Julie M. , Smith Lynette M. , Armitage James O. , Mayer R. Aeryn , Pike Brian L. , Collins Francis S. , Hacia Joseph G.
Published: April 2003

Mutations have been described in the ataxia telangiectasia mutated (
ATM
) gene in small numbers of cases of lymphoid neoplasia. However, surveys of the
ATM
mutation status in lymphoma have been limited due to the large size (62 exons) and complex mutational spectrum of this gene. We have used microarray-based assays with 250,000 oligonucleotides to screen lymphomas from 120 patients for all possible
ATM
coding and splice junction mutations. The subtypes included were diffuse large B cell, mantle cell, immunoblastic large B cell, follicular, posttransplant lymphoproliferative disorder, and peripheral T cell lymphoma. We found the highest percentage of
ATM
mutations within the mantle cell (MCL) subtype (43%, 12 of 28 cases), followed by a lower level (10% of cases) in the other subtypes. A frame-shift
ATM
mutation was found in one peripheral T cell lymphoma patient. In six MCL cases examined, four
ATM
variants were due to somatic mutation in the tumor cells whereas two others seemed to be germ-line in origin. There was no difference in
p53
mutation status in the
ATM
mutant and wild-type groups of MCL. There was no statistically significant difference in the median overall survival of patients with wild-type vs. mutated
ATM
in MCL. Additional mutational and functional analyses are needed to determine whether
ATM
mutations contribute to the development and progression of MCL or are just the consequence of genomic instability in MCL.
Identification of a series of CCK-2 receptor nonpeptide agonists: Sensitivity to stereochemistry and a receptor point mutation
Authors: Kopin Alan S. , McBride Edward W. , Chen Ci , Freidinger Roger M. , Chen Duan , Zhao Chun-Mei , Beinborn Martin
Published: April 2003

The search for small-molecule drugs that act at peptide hormone receptors has resulted in the identification of a wide variety of antagonists. In contrast, the discovery of nonpeptide agonists has been far more elusive. We have used a constitutively active mutant of the cholecystokinin 2 receptor (CCK-2R) as a sensitive screen to detect ligand activity. Functional assessment of structural analogs of the prototype CCK-2R antagonist, L-365,260 [3
R
-
N
- (2,3-dihydro-1-methyl-2-oxo-5-phenyl-1
H
-1,4-benzodiazepin-3-yl)-
N
′-(3-methylphenyl)urea], resulted in the identification of a series of agonists. Each of the active molecules is an
S
enantiomer, whereas the corresponding
R
stereoisomers have little or no activity. Further
in vitro
and
in vivo
assessment at the wild-type receptor indicated that efficacy of the two most active ligands approached that of the endogenous hormone. The function of selected
R
and
S
enantiomers was differentially sensitive to a point mutation, N353L, within the putative CCK-2R ligand pocket. The results of this study highlight the potential of constitutively active receptors as drug screening tools and the interdependence of ligand stereochemistry and receptor conformation in defining drug efficacy.
Band 3 is a host receptor binding merozoite surface protein 1 during thePlasmodium falciparuminvasion of erythrocytes
Authors: Goel Vikas K. , Li Xuerong , Chen Huiqing , Liu Shih-Chun , Chishti Athar H. , Oh Steven S.
Published: April 2003

We report the molecular identification of a sialic acid-independent host–parasite interaction in thePlasmodium falciparummalaria parasite invasion of RBCs. Two nonglycosylated exofacial regions of human band 3 in the RBC membrane were identified as a crucial host receptor binding the C-terminal processing products of merozoite surface protein 1 (MSP1). Peptides derived from the receptor region of band 3 inhibited the invasion of RBCs byP. falciparum. A major segment of the band 3 receptor (5ABC) bound to native MSP142and blocked the interaction of native MSP142with intact RBCsin vitro. Recombinant MSP119(the C-terminal domain of MSP142) bound to 5ABC as well as RBCs. The binding of both native MSP142and recombinant MSP119was not affected by the neuraminidase treatment of RBCs, but sensitive to chymotrypsin treatment. In addition, recombinant MSP138showed similar interactions with the band 3 receptor and RBCs, although the interaction was relatively weak. These findings suggest that the chymotrypsin-sensitive MSP1–band 3 interaction plays a role in a sialic acid-independent invasion pathway and reveal the function of MSP1 in thePlasmodiuminvasion of RBCs.
Endonuclease cleavage of blocked replication forks: An indirect pathway of DNA damage from antitumor drug–topoisomerase complexes
Authors: Hong George , Kreuzer Kenneth N.
Published: April 2003

The cytotoxicity of several important antitumor drugs depends on formation of the covalent topoisomerase–DNA cleavage complex. However, cellular processes such as DNA replication are necessary to convert the cleavage complex into a cytotoxic lesion, but the molecular mechanism of this conversion and the precise nature of the cytotoxic lesion are unknown. Using a bacteriophage T4 model system, we have previously shown that antitumor drug-induced cleavage complexes block replication forks
in vivo
. In this report, we show that these blocked forks can be cleaved by T4 endonuclease VII to create overt DNA breaks. The accumulation of blocked forks increased in endonuclease VII-deficient infections, suggesting that endonuclease cleavage contributes to fork processing
in vivo
. Furthermore, purified endonuclease VII cleaved the blocked forks
in vitro
close to the branch points. These results suggest that an indirect pathway of branched-DNA cleavage contributes to the cytotoxicity of antitumor drugs that target DNA topoisomerases.
Cyclooxygenase-2 is instrumental in Parkinson's disease neurodegeneration
Authors: Teismann Peter , Tieu Kim , Choi Dong-Kug , Wu Du-Chu , Naini Ali , Hunot Stéphane , Vila Miquel , Jackson-Lewis Vernice , Przedborski Serge
Published: April 2003

Parkinson's disease (PD) is a neurodegenerative disorder of uncertain pathogenesis characterized by the loss of the nigrostriatal dopaminergic neurons, which can be modeled by the neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). Increased expression of cyclooxygenase type 2 (COX-2) and production of prostaglandin E
2
have been implicated in neurodegeneration in several pathological settings. Here we show that COX-2, the rate-limiting enzyme in prostaglandin E
2
synthesis, is up-regulated in brain dopaminergic neurons of both PD and MPTP mice. COX-2 induction occurs through a JNK/c-Jun-dependent mechanism after MPTP administration. We demonstrate that targeting COX-2 does not protect against MPTP-induced dopaminergic neurodegeneration by mitigating inflammation. Instead, we provide evidence that COX-2 inhibition prevents the formation of the oxidant species dopamine-quinone, which has been implicated in the pathogenesis of PD. This study supports a critical role for COX-2 in both the pathogenesis and selectivity of the PD neurodegenerative process. Because of the safety record of the COX-2 inhibitors, and their ability to penetrate the blood–brain barrier, these drugs may be therapies for PD.
The molecular basis for the chemical denaturation of proteins by urea
Authors: Bennion Brian J. , Daggett Valerie
Published: April 2003

Molecular dynamics simulations of the protein chymotrypsin inhibitor 2 in 8 M urea at 60°C were undertaken to investigate the molecular basis of chemical denaturation. The protein unfolded rapidly under these conditions, but it retained its native structure in a control simulation in water at the same temperature. The overall process of unfolding in urea was similar to that observed in thermal denaturation simulations above the protein's
T
m
of 75°C. The first step in unfolding was expansion of the hydrophobic core. Then, the core was solvated by water and later by urea. The denatured structures in both urea and at high temperature contained residual native helical structure, whereas the β-structure was completely disrupted. The average residence time for urea around hydrophilic groups was six times greater than around hydrophobic residues and in all cases greater than the corresponding water residence times. Water self-diffusion was reduced 40% in 8 M urea. Urea altered water structure and dynamics, thereby diminishing the hydrophobic effect and encouraging solvation of hydrophobic groups. In addition, through urea's weakening of water structure, water became free to compete with intraprotein interactions. Urea also interacted directly with polar residues and the peptide backbone, thereby stabilizing nonnative conformations. These simulations suggest that urea denatures proteins via both direct and indirect mechanisms.
Directional selection has shaped the oral jaws of Lake Malawi cichlid fishes
Authors: Albertson R. Craig , Streelman J. Todd , Kocher Thomas D.
Published: April 2003

East African cichlid fishes represent one of the most striking examples of rapid and convergent evolutionary radiation among vertebrates. Models of ecological speciation would suggest that functional divergence in feeding morphology has contributed to the origin and maintenance of cichlid species diversity. However, definitive evidence for the action of natural selection has been missing. Here we use quantitative genetics to identify regions of the cichlid genome responsible for functionally important shape differences in the oral jaw apparatus. The consistent direction of effects for individual quantitative trait loci suggest that cichlid jaws and teeth evolved in response to strong, divergent selection. Moreover, several chromosomal regions contain a disproportionate number of quantitative trait loci, indicating a prominent role for pleiotropy or genetic linkage in the divergence of this character complex. Of particular interest are genomic intervals with concerted effects on both the length and height of the lower jaw. Coordinated changes in this area of the oral jaw apparatus are predicted to have direct consequences for the speed and strength of jaw movement. Taken together, our results imply that the rapid and replicative nature of cichlid trophic evolution is the result of directional selection on chromosomal packages that encode functionally linked aspects of the craniofacial skeleton.
On schemes of combinatorial transcription logic
Authors: Buchler Nicolas E. , Gerland Ulrich , Hwa Terence
Published: April 2003

Cells receive a wide variety of cellular and environmental signals, which are often processed combinatorially to generate specific genetic responses. Here we explore theoretically the potentials and limitations of combinatorial signal integration at the level of cis-regulatory transcription control. Our analysis suggests that many complex transcription-control functions of the type encountered in higher eukaryotes are already implementable within the much simpler bacterial transcription system. Using a quantitative model of bacterial transcription and invoking only specific protein–DNA interaction and weak glue-like interaction between regulatory proteins, we show explicit schemes to implement regulatory logic functions of increasing complexity by appropriately selecting the strengths and arranging the relative positions of the relevant protein-binding DNA sequences in the cis-regulatory region. The architectures that emerge are naturally modular and evolvable. Our results suggest that the transcription regulatory apparatus is a “programmable” computing machine, belonging formally to the class of Boltzmann machines. Crucial to our results is the ability to regulate gene expression at a distance. In bacteria, this can be achieved for isolated genes via DNA looping controlled by the dimerization of DNA-bound proteins. However, if adopted extensively in the genome, long-distance interaction can cause unintentional intergenic cross talk, a detrimental side effect difficult to overcome by the known bacterial transcription-regulation systems. This may be a key factor limiting the genome-wide adoption of complex transcription control in bacteria. Implications of our findings for combinatorial transcription control in eukaryotes are discussed.
Cell division pattern influences gene expression in the shoot apical meristem
Authors: Wyrzykowska Joanna , Fleming Andrew
Published: April 2003

The shoot apical meristem of angiosperms shows a highly conserved cellular architecture in which a change of cell division orientation correlates with early events of leaf initiation. However, the causal role of this altered cellular parameter in leaf formation is debatable. We have used the dynamin-like protein phragmoplastin as a tool to modify the pattern of cell division within the apical meristem. Taking a microinduction approach, we show that local alteration in cell division orientation is not sufficient to induce morphogenesis in the meristem. Surprisingly, an altered cell division pattern did lead to an altered pattern of expression of genes implicated in aspects of leaf formation. Our data identify inducible expression of phragmoplastin as a tool to manipulate cell division pattern. Furthermore, they indicate that a mechanism exists by which cells in the meristem can respond at the level of gene expression to altered parameters of cell division. These data are discussed in the context of a model linking leaf morphogenesis and differentiation.
Chaperone action in the posttranslational topological reorientation of the hepatitis B virus large envelope protein: Implications for translocational regulation
Authors: Lambert Carsten , Prange Reinhild
Published: April 2003

The large L envelope protein of the hepatitis B virus utilizes a new folding pathway to acquire a dual transmembrane topology in the endoplasmic reticulum (ER). The process involves cotranslational membrane integration and subsequent posttranslational translocation of its preS subdomain into the ER. Here, we demonstrate that the conformational and functional heterogeneity of L depends on the action of molecular chaperones. Using coimmunoprecipitation, we observed specific interactions between L and the cytosolic Hsc70, in conjunction with Hsp40, and between L and the ER-resident BiP in mammalian cells. Complex formation between L and Hsc70 was abolished when preS translocation was artificially switched to a cotranslational mode, implicating Hsc70 to act as a preS holding and folding catalyst that controls partial preS posttranslocation. The functional role of Hsc70 in L topogenesis was confirmed through modulation of its
in vivo
activity by overexpressing its co-chaperones Hip and Bag-1. Overexpression of the Hsc70-stimulating molecule Hip led to increased entrapping of preS on the cytosolic ER face and hence to a decrease in preS posttranslocation, whereas the negative regulator Bag-1 had the opposite effects. Furthermore, Hip-mediated Hsc70 activation impaired virus production in hepatitis B virus-replicating hepatoma cells, likely due to the improper topological reorientation of L. Together, these results indicate that translocational regulation of protein topology by chaperones provides a means of generating structural and functional diversity. They also hint to the dynamic nature of the mammalian ER translocation machinery in handling co- and posttranslational substrates.
Cryptic species of fig-pollinating wasps: Implications for the evolution of the fig–wasp mutualism, sex allocation, and precision of adaptation
Authors: Molbo Drude , Machado Carlos A. , Sevenster Jan G. , Keller Laurent , Herre Edward Allen
Published: May 2003

Fig-pollinating wasps have provided model systems for developing and testing theories of the evolution of mutualism, sex allocation, and precision of adaptation. With few exceptions, previous studies have assumed one species of pollinator wasp per host fig species. Here we report genetic data demonstrating the coexistence of previously undetected cryptic fig wasp species in at least half of the host fig species surveyed. The substantial mitochondrial sequence differences (4.2–6.1%) imply old divergences (≈1.5–5.1 million years ago) among these species. Furthermore, some cryptic species pairs seem to be sister taxa, whereas others clearly are not, indicating both long-term coexistence on shared hosts and the colonization of novel fig species. These findings undermine the prevalent notion of strict one-to-one specificity between cospeciating figs and their pollinators, thereby challenging existing theory concerning the evolution and stability of mutualisms. Moreover, the incorporation of the genetic information significantly improves the fit of the observed sex ratios to predictions of local mate-competition theory, further strengthening support for sex allocation theory and the precision of adaptation.