bluuebunny/crossref_metadata_2025 huggingface dataset:
Passive Immunisation with IgG Endotoxin Core Antibody Hyperimmune Fresh Frozen Plasma
Authors: Hamilton‐Davies C. , Barclay G.R. , Murphy W.G. , Machin S.J. , Webb A.R.
Published: May 2003

Postoperative complications have been associated with low pre‐operative endotoxin core antibody (EndoCAb) status. Seventy‐three patients scheduled to undergo elective cardiac surgery were screened for IgG EndoCAb levels 1 week prior to admission. Of these 73 patients, 39 had levels below the median of a healthy population (100 MU = median units/ml). Only 6 received an infusion of IgG EndoCAb hyperimmune fresh frozen plasma (FFP) due to surgical rescheduling or unavailability of compatible hyperimmune FFP. The 6 patients demonstrated significant elevation (p < 0.0001) of their IgG EndoCAb levels from a mean level of 43 MU/ml (range: 26–75) to a posttransfusion mean of 267 MU/ml (range: 227–300), which persisted until at least immediately prior to the operative period. We conclude that passive immunisation with EndoCAb hyperimmune FFP is possible prior to elective surgery and that the kinetic profile of such an administration will allow the FFP to be given the night prior to surgery and still maintain significantly elevated levels for surgery planned the following morning.
Two Missense Mutations of H Type α(1,2)Fucosyltransferase Gene (FUT1)Responsible for Para‐Bombay Phenotype
Authors: Wang Baojie , Koda Yoshiro , Soejima Mikiko , Kimura Hiroshi
Published: May 2003

Background and objectives: Rare individuals (Bombay and para‐Bombay phenotypes) fail to express the A, B and H antigens on erythrocyte membranes because of a lack in the H gene (FUT1)‐encoded α(1,2)fucosyltransferase activity. In this study, we have found a para‐Bombay individual (Bmh) who expressed B and H antigens in saliva but not on red blood cells. The FUT1 alleles of this person contained two single base changes (T460C and G1042A) in the coding region relative to the wild type allele. These substitutions may result in changes in two amino acid residues (Y154H and E348K). Materials and methods: Since the T460C and G1042A mutations destroy endonuclease RsaI and AvaI sites, respectively, we tested for these mutations using PCR‐RFLP. Results: Our findings indicated that this para‐Bombay person was homozygous for the T460C and G1042A mutations, and that neither of these mutations was found in 136 randomly selected Japanese individuals. The measurement of the α(1,2)fucosyltransferase activity after transient expression of the FUT1 alleles in COS‐7 cells indicated that the H‐deficient allele‐encoded enzyme had no detectable activity. Moreover, transfection by chimera FUT1 allele contains only the T460C mutation, or only the G1042A mutation, and yielded 1.0 or 9.3%, respectively, of the activities compared to transfection by the wild type allele. Conclusions: These results suggest that the two mutations in combination are responsible for the inactivation of the FUT1‐encoded enzyme activity.
Quantitative Analysis of Myelinated Axons of Commissural Fibers in the Rat Brain
Authors: Sargon M. F. , Mas N. , Şenan S. , Özdemi̇r B. , Çeli̇k H. H. , Cumhur M.
Published: May 2003

In this study, the myelinated axons of the rostrum, genu, truncus and splenium parts of the corpus callosum and of the anterior, posterior and habenular commissures were counted in the rat brain by using a camera lucida. The numerical densities of these axons were compared with each other by means of quantitative analytical statistical methods. In parts of the corpus callosum, a statistically significant difference was found between the rostrum and genu, rostrum and truncus, rostrum and the splenium, genu and truncus, and the genu and splenium. However, no statistically significant difference was found between the truncus and splenium. When comparing the number of myelinated axons of the anterior, posterior and habenular commissures, statistically significant differences were found between the anterior and posterior commissures, and between the anterior and habenular commissures. No statistically significant difference was found between the posterior and habenular commissures. Small sized myelinated axons were present in all parts of the corpus callosum and in the anterior commissure. However, a heterogeneous distribution of myelinated axons was present in the posterior and habenular commissures.
Development of the Glandular Epithelium of the Bovine Parotid Gland during Ontogenesis
Authors: Eisenbrückner A. , Fink C. , Kressin M.
Published: May 2003

The development of the parotid gland was examined in 36 bovine embryos and foetuses with a crown‐rump‐length (CRL) from 28 up to 1000 mm by light, transmission electron microscopical and actin‐immunohistochemical methods.

The anlage of the parotid gland in an embryo with 28 mm CRL can be found at the lateral angle of the primitive oral cavity as a local thickening of the epithelium. During the second month, the differentiation of primary ducts and endbuds starts and a lumen develops in the primary ducts. At the end of the second month a lumen appears in the terminal endbuds. In the immature endpiece cells first secretory granules can be seen from a CRL of 240 mm. In the third month differentiation between intra‐ and inter‐lobular ducts is possible.

Immature myoepithelial cells present as a basal layer of flattened cells between the epithelial cells and the basement membrane at the end of the second month. During further development they increase in number, become more flattened and form long cellular processes. At the end of the fourth month isolated actin filament bundles are formed, which were also detected by an antibody against smooth muscle actin. The actin filaments condense continuously until they fill the cell processes completely at the end of foetal development.
Ofuji papuloerythroderma
Authors: Aste N , Fumo G , Conti B , Biggio P
Published: May 2003

Ofuji papuloerythrodema is a rare type of dermatosis marked by an erythrodermic manifestation which is intensely pruritic and results from the coalescing of brownish papules which in the main do not involve the great folds. The authors present the case history of a 72‐year‐old female, whose dermatosis appeared 8 years after the diagnosis of malignant lymphocitic lymphoma. The Authors describe the clinical and histopathological picture, illustrating the laboratory results and stressing the importance of the association of the two pathologies in a possible nosological overview.
An examination of the needs of parents of hospitalized children: comparing parents’ and staff's perceptions
Authors: Shields Linda , Kristensson‐Hallström Inger , O'Callaghan Michael
Published: May 2003

The needs of parents of hospitalized children have received some attention in the health literature, but few studies have compared parents’ perceptions of needs with staff's ideas about parents’ needs. The aim of this study was to examine differences between the perceptions of the needs of parents of hospitalized children held by staff – nurses, doctors and allied health staff, and parents in a 150‐bed paediatric hospital in Sweden. The convenience sample comprised 132 staff – nurses, doctors and allied health staff and 115 parents of children admitted to all the wards except intensive care. Kristjánsdóttir's “needs of parents of hospitalized children” questionnaire (NPQ) was the instrument of choice and was modified slightly for use with staff. Results indicated significant differences in perceptions of the importance of different needs of parents, of how well they were being met in the hospital and how much help the parents needed to have them filled. Differences between parents’ and staff's perceptions of the importance of parental needs were found in areas relating to psychosocial needs, but in general, in that hospital, the needs were being adequately met. The main differences between staff's and parents’ results were in the degree of independence shown by parents in requiring help to have their needs met. This demonstrates either that parents are much more independent than appraised by staff, or, that parents are sometimes unaware of the level of assistance available.
Papillomavirus and disease in humans and animals
Authors: Campo M. S.
Published: May 2003

Papillomavirus (PV) induces exophytic lesions (papillomas, warts) and flat lesions (flat warts, cervical intraepithelial neoplasia) in cutaneous and mucosal epithelia. The lesions are usually benign and generally regress without eliciting any serious clinical problems in a host but occasionally persist. Persistent lesions can be debilitating and can also provide a focus for malignant transformation to squamous cell carcinoma, particularly in the presence of environmental or genetic cofactors. This has been experimentally demonstrated in animals, particularly in cattle, where bovine PV (BPV)‐induced papillomas progress to cancer of the upper gastrointestinal (GI) tract and the urinary bladder in animals exposed to bracken fern in the pasture, and in rabbits, where the progression of skin papillomas to squamous cell carcinoma depends on a particular variant of cottontail rabbit PV (CRPV) and on the major histocompatibility complex (MHC) class II haplotype of the animal. In this review, various aspects of the biology of BPV and CRPV are described and compared with those of human PV, including viral genome structure, regulation of transcription of the viral oncogenes, function of the viral oncoproteins, co‐operation between virus and cofactors, virus latency, immunity and vaccination.
Randomized Trial Comparing Albumin and Saline in the Prevention of Paracentesis–Induced Circulatory Dysfunction in Cirrhotic Patients With Ascites
Authors: Sola-Vera Javier , Miñana Josep , Ricart Elena , Planella Montserrat , González Begoña , Torras Xavier , Rodríguez Jose , Such José , Pascual Sonia , Soriano Germán , Pérez-Mateo Miguel , Guarner Carlos
Published: May 2003

Paracentesis–induced circulatory dysfunction (PICD) is a recently described complication that can be prevented with the administration of plasma expanders. The aim of this study was to compare the efficacy of saline versus albumin in the prevention of PICD. Patients were randomized to receive albumin or saline after total paracentesis. Patients readmitted as a consequence of a second episode of tense ascites were treated with total paracentesis and the alternative plasma expander. After randomization, 35 patients received saline and 37 received albumin. Twenty–one patients were readmitted for tense ascites and treated with the alternative expander. Significant increases in plasma renin activity (PRA) were found 24 hours and 6 days after paracentesis when saline was used (baseline, 5.6 ± 5.7; 24 hours, 7.6 ± 6.9; 6 days, 8.5 ± 8.0 ng · mL-1 · hr-1; P < .05 and P < .01 vs. baseline, respectively), whereas no significant changes were observed with albumin. The incidence of PICD was significantly higher in the saline group versus the albumin group (33.3% vs. 11.4%, respectively; P = .03). However, no significant differences were found when less than 6 L of ascitic fluid was evacuated (6.7% vs. 5.6% in the saline and albumin groups, respectively; P = .9). Similar results were observed when analyzing patients who received 2 consecutive paracentesis (i.e., a significant increase in PRA after saline [P < .01] without significant variations after albumin). In conclusion, albumin is more effective than saline in the prevention of PICD. Saline is a valid alternative to albumin when less than 6 L of ascitic fluid is evacuated.
The Normal Liver Harbors the Vitamin D Nuclear Receptor in Nonparenchymal and Biliary Epithelial Cells
Authors: Gascon-Barré Marielle , Demers Christian , Mirshahi Ali , Néron Stéphane , Zalzal Sylvia , Nanci Antonio
Published: May 2003

The liver is generally considered negative for the vitamin D nuclear receptor (VDRn), even though several studies have shown significant effects of 1,25–dihydroxyvitamin D3 (1,25(OH)2D3) on liver cell physiology. The low abundance of VDRn in the liver led us to propose that hepatocytes (the largest hepatic cell population) were most likely negative for the receptor, whereas the small hepatic sinusoidal and ductular cell populations that contain cell types known to express VDRn in other tissues should express the receptor. Using freshly isolated cells from normal livers as well as biliary and epithelial hepatic cell lines, our data show that the human, rat, and mouse hepatocytes express very low VDRn messenger RNA (mRNA) and protein levels. In contrast, sinusoidal endothelial, Kupffer, and stellate cells of normal rat livers as well as the mouse biliary cell line BDC and rat hepatic neonatal epithelial SD6 cells clearly expressed both VDRn mRNA and protein. In addition, specimens of human hepatocarcinoma as well as intrahepatic colon adenocarcinoma metastases were also found to express the VDRn
gene transcript. Kupffer, stellate, and endothelial cells responded to 1,25(OH)2D3 by a significant increase in the CYP24, indicating that the VDRn is fully functional in these cells. In conclusion, selective hepatic cell populations are targets for the vitamin D endocrine/paracrine/intracrine system.
Troglitazone Induces P27 Kip1 –Associated Cell–Cycle Arrest Through Down–Regulating Skp2 in Human Hepatoma Cells
Authors: Koga Hironori , Harada Masaru , Ohtsubo Motoaki , Shishido Shoichiro , Kumemura Hiroto , Hanada Shinichiro , Taniguchi Eitaro , Yamashita Katsumi , Kumashiro Ryukichi , Ueno Takato , Sata Michio
Published: May 2003

Increasing evidence has confirmed that ligands for peroxisome proliferator–activated receptor γ (PPARγ) exhibit antitumoral effects through inhibition of cell proliferation and induction of cell differentiation in several malignant neoplasms. Recently, we have documented the accumulation of a cyclin–dependent kinase inhibitor, p27
Kip1
, as well as an unexpected accumulation in cyclin E in G1–arrested human hepatoma cells treated with the PPARγ ligand troglitazone. Simultaneous accumulations in both p27
Kip1
and cyclin E are known to be characteristic phenotypes in cells derived from mice lacking Skp2, an F–box protein component of the SCF ubiquitin–ligase complex. Thus, the aim of the present study was to assess whether Skp2 might be involved in the down–regulation of p27
Kip1
in troglitazone–treated human hepatoma cells. A striking decrease in Skp2 expression and a reciprocal increase in p27
Kip1
expression were found in troglitazone–treated hepatoma cells but not in those cells treated with other PPARγ ligands such as pioglitazone and ciglitazone. Quantitative real–time RT–PCR analysis showed that troglitazone down–regulated Skp2 at the mRNA levels. Consistently, ectopic overexpression in Skp2 brought resistance to troglitazone, resulting in a decreased population of arrested cells at the G1 phase compared with that in the mock–transfected cells. In surgically resected hepatocellular carcinoma (HCC) tissue, an increased expression in Skp2 was found in both the moderately differentiated HCCs and the poorly differentiated HCCs. In conclusion, troglitazone attenuated Skp2 expression, thereby promoting p27
Kip1
accumulation in human hepatoma cells. This therapeutic potential of the ligand may lead to new cell–cycle–based antitumor strategies for advanced HCCs.
Nonalcoholic Steatohepatitis: Summary of An Aasld Single Topic Conference
Authors: Neuschwander-Tetri Brent A. , Caldwell Stephen H.
Published: May 2003

Fatty liver disease that develops in the absence of alcohol abuse is recognized increasingly as a major health burden. This report summarizes the presentations and discussions at a Single Topic Conference held September 20–22, 2002, and sponsored by the American Association for the Study of Liver Diseases. The conference focused on fatty liver disorders. Estimates based on imaging and autopsy studies suggest that about 20% to 30% of adults in the United States and other Western countries have excess fat accumulation in the liver. About 10% of these individuals, or fully 2% to 3% of adults, are estimated to meet current diagnostic criteria for nonalcoholic steatohepatitis (NASH). Sustained liver injury leads to progressive fibrosis and cirrhosis in a fraction, possibly up to one third, of those with NASH, and NASH may be a cause of cryptogenic cirrhosis. NASH is now a significant health issue for obese children as well, leading to cirrhosis in some. The diagnostic criteria for NASH continue to evolve and rely on the histologic findings of steatosis, hepatocellular injury (ballooning, Mallory bodies), and the pattern of fibrosis. Generally recognized indications for biopsy include establishing the diagnosis and staging of the injury, but strict guidelines do not exist. Liver enzymes are insensitive and cannot be used reliably to confirm the diagnosis or stage the extent of fibrosis. Older age, obesity, and diabetes are predictive of fibrosis. The pathogenesis of NASH is multifactorial. Insulin resistance may be an important factor in the accumulation of hepatocellular fat, whereas excess intracellular fatty acids, oxidant stress, adenosine triphosphate (ATP) depletion, and mitochondrial dysfunction may be important causes of hepatocellular injury in the steatotic liver. Efforts are underway to refine the role of insulin resistance in NASH and determine whether improving insulin sensitivity pharmacologically is an effective treatment. An altered lifestyle may be a more effective means of improving insulin sensitivity. The research agenda for the future includes establishing the role of insulin resistance and abnormal lipoprotein metabolism in NASH, determining the pathogenesis of cellular injury, defining predisposing genetic abnormalities, identifying better noninvasive predictors of disease, and defining effective therapy.
Expanding tRNA recognition of a tRNA synthetase by a single amino acid change
Authors: Feng Liang , Tumbula-Hansen Debra , Toogood Helen , Söll Dieter
Published: May 2003

Aspartyl-tRNA synthetase (AspRS) occurs in two types: the discriminating enzyme (D-AspRS) forms only Asp-tRNA
Asp
, whereas the nondiscriminating enzyme (ND-AspRS) also synthesizes Asp-tRNA
Asn
, which is a required intermediate for protein synthesis in many organisms. We attempted to expand the tRNA recognition of the discriminating
Thermococcus kodakaraensis
AspRS to that of a ND-AspRS by
in vitro
mutagenesis. An alignment of 26 archaeal AspRS proteins revealed two positions (26 and 85 in the
T. kodakaraensis
sequence) whose amino acid identity changes according to the enzymes' tRNA specificity. In their anticodon-binding domain, D-AspRS proteins contain W26 (or Q26) and K85, compared with H26 and P85 in the ND-AspRSs.
T. kodakaraensis
AspRS gained the ability to form Asp-tRNA
Asn
in vitro
when the W26H or K85P changes were introduced independently or in combination. In the aminoacylation of tRNA
Asn
or tRNA
Asp
transcripts, the mutant enzymes displayed at least a 100- to 500-fold change in tRNA specificity, as judged by the ratio of the
k
cat
/
K
m
values of Asp-tRNA
Asp
vs. Asp-tRNA
Asn
formation. That
T. kodakaraensis
mutant AspRSs mischarge tRNA
Asn
was also manifested in the higher level (1.7%) of aspartylation of unfractionated
Pyrococcus
tRNA compared with that achieved by the wild-type enzyme (0.9%). Northern blot analysis of the Asp-tRNA separated by acid/urea gel electrophoresis confirmed the
in vitro
synthesis of Asp-tRNA
Asn
. A structure-based model points to a direct interaction of K85 in
T. kodakaraensis
AspRS with the anticodon nucleotide C36 of tRNA
Asp
. Thus, a switch between D-AspRS and ND-AspRS enzymes could have evolved with only limited amino acid changes.
Parkin
, a gene implicated in autosomal recessive juvenile parkinsonism, is a candidate tumor suppressor gene on chromosome 6q25–q27
Authors: Cesari Rossano , Martin Eric S. , Calin George A. , Pentimalli Francesca , Bichi Roberta , McAdams Holly , Trapasso Francesco , Drusco Alessandra , Shimizu Masayoshi , Masciullo Valeria , d'Andrilli Giuseppina , Scambia Giovanni , Picchio Maria Cristina , Alder Hansjuerg , Godwin Andrew K. , Croce Carlo M.
Published: May 2003

In an effort to identify tumor suppressor gene(s) associated with the frequent loss of heterozygosity observed on chromosome 6q25–q27, we constructed a contig derived from the sequences of bacterial artificial chromosome/P1 bacteriophage artificial chromosome clones defined by the genetic interval D6S1581–D6S1579–D6S305–D6S1599–D6S1008. Sequence analysis of this contig found it to contain eight known genes, including the complete genomic structure of the
Parkin
gene. Loss of heterozygosity (LOH) analysis of 40 malignant breast and ovarian tumors identified a common minimal region of loss, including the markers D6S305 (50%) and D6S1599 (32%). Both loci exhibited the highest frequencies of LOH in this study and are each located within the
Parkin
genomic structure. Whereas mutation analysis revealed no missense substitutions, expression of the
Parkin
gene appeared to be down-regulated or absent in the tumor biopsies and tumor cell lines examined. In addition, the identification of two truncating deletions in 3 of 20 ovarian tumor samples, as well as homozygous deletion of exon 2 in the lung adenocarcinoma cell lines Calu-3 and H-1573, supports the hypothesis that hemizygous or homozygous deletions are responsible for the abnormal expression of
Parkin
in these samples. These data suggest that the LOH observed at chromosome 6q25–q26 may contribute to the initiation and/or progression of cancer by inactivating or reducing the expression of the
Parkin
gene. Because
Parkin
maps to
FRA6E
, one of the most active common fragile sites in the human genome, it represents another example of a large tumor suppressor gene, like
FHIT
and
WWOX
, located at a common fragile site.
Dual-frequency 2D-IR spectroscopy heterodyned photon echo of the peptide bond
Authors: Rubtsov Igor V. , Wang Jianping , Hochstrasser Robin M.
Published: May 2003

The structure fluctuations of the peptide bond interacting with solvent are examined through the coupling and correlations of the frequency distributions of amide I and amide II transitions. The fluctuations of the two modes are anticorrelated as a result of the solvent-induced changes in the mixing of the dominant valence-bond structures of the peptide. Significant anharmonic coupling of the two modes is seen. The results are the application of a new approach to two-dimensional infrared (2D-IR) spectroscopy in which the pulse sequences used to produce the vibrational echoes incorporate two frequencies. This dual-frequency arrangement greatly extends the capabilities of 2D-IR spectroscopy by allowing the coupling between widely separated modes to be characterized in analogy with heteronuclear NMR. The experiment exposes the cross peaks, representing the mode coupling, free of the interference of the strong diagonal peaks that typically dominate 2D-IR spectroscopy. The alignment and dephasing of coupled transitions, in this example the amide I and amide II transition dipoles, is also determined by these experiments.
Adaptive evolution of cytochrome
c
oxidase subunit VIII in anthropoid primates
Authors: Goldberg Allon , Wildman Derek E. , Schmidt Timothy R. , Hüttemann Maik , Goodman Morris , Weiss Mark L. , Grossman Lawrence I.
Published: May 2003

Cytochrome
c
oxidase (COX) is a 13-subunit protein complex that catalyzes the last step in mitochondrial electron transfer in mammals. Of the 10 subunits encoded by nuclear DNA (three are mtDNA products), some are expressed as tissue- and/or development-specific isoforms. For COX subunit VIII, previous work showed that expression of the contractile muscle-specific isoform gene,
COX8H
, is absent in humans and Old World monkeys, and the other isoform gene,
COX8L
, is expressed ubiquitously. Here, we show that
COX8H
is transcribed in most primate clades, but its expression is absent in catarrhines, that is, in Old World monkeys and hominids (apes, including humans), having become a pseudogene in the stem of the catarrhines. The ubiquitously expressed isoform,
COX8L
, underwent nonsynonymous rate acceleration and elevation in the ratio of nonsynonymous/synonymous changes in the stem of anthropoid primates (New World monkeys and catarrhines), possibly setting the stage for loss of the heart-type (H) isoform. The most rapidly evolving region of VIII-L is one that interacts with COX I, suggesting that the changes are functionally coadaptive. Because accelerated rates of nonsynonymous substitutions in anthropoids such as observed for
COX8L
are also shown by genes for at least 13 other electron transport chain components, these encoded amino acid replacements may be viewed as part of a series of coadaptive changes that optimized the anthropoid biochemical machinery for aerobic energy metabolism. We argue that these changes were linked to the evolution of an expanded neocortex in anthropoid primates.
Iron chelators modulate the fusogenic properties of
Salmonella
-containing phagosomes
Authors: Jabado Nada , Cuellar-Mata Patricia , Grinstein Sergio , Gros Philippe
Published: May 2003

In macrophages, the divalent cations transporter Nramp1 is recruited from the lysosomal compartment to the membrane of phagosomes formed in these cells.
Nramp1
mutations cause susceptibility to infection with intracellular pathogens such as
Salmonella
and
Mycobacterium
. Intracellular survival of
Salmonella
involves segregation in an endomembrane compartment (
Salmonella
-containing vacuole, SCV) that remains negative for the mannose-6-phosphate receptor (M6PR) and that is inaccessible to the endocytic pathway. Expression of Nramp1 at the membrane of SCVs stimulates both acquisition of M6PR and accessibility to newly formed endosomes. The possible role of Nramp1-mediated iron transport on SCV maturation was investigated with membrane-permeant iron chelators. Pretreatment of primary macrophages from
Nramp1
mutant mice or of RAW264.7 macrophages (from BALB/c mice bearing an

Nramp1
D169

-deficient allele) with either desferrioxamine or salicylaldehyde isocotinoyl hydrazone restored recruitment of M6PR and delivery of the fluid phase marker rhodamine dextran to SCVs to levels similar to those seen in macrophages expressing WT
Nramp1
. The effect was specific and dose-dependent and could be abrogated by preincubation with excess iron. These data suggest that Nramp1-mediated deprivation of iron and possibly of other divalent metals in macrophages antagonizes the ability of
Salmonella
to alter phagosome maturation.
Identification and characterization of a G protein-coupled receptor for the neuropeptide proctolin in
Drosophila
melanogaster
Authors: Johnson Erik C. , Garczynski Stephen F. , Park Dongkook , Crim Joe W. , Nässel Dick R. , Taghert Paul H.
Published: May 2003

Proctolin is a bioactive neuropeptide that modulates interneuronal and neuromuscular synaptic transmission in a wide variety of arthropods. We present several lines of evidence to propose that the orphan G protein-coupled receptor
CG6986
of
Drosophila
is a proctolin receptor. When expressed in mammalian cells,
CG6986
confers second messenger activation after proctolin application, with an EC
50
of 0.3 nM. In competition-based studies, the
CG6986
receptor binds proctolin with high affinity (IC
50
= 4 nM). By microarray analysis,
CG6986
transcript is consistently detected in head mRNA of different genotypes, and under different environmental conditions. By blot analysis, anti-
CG6986
antibodies detect a band in tissue homogenates similar to the predicted size of the protein. Proctolin receptor immunosignals are found in the hindgut, heart, and in distinct neuronal populations of the CNS; such patterns correlate with previous demonstrations of proctolin biological activity, and in several instances, with areas of proctolin peptide immunosignals. The identification of a
bona fide
proctolin receptor provides the basis for a mechanistic analysis of this critical synaptic modulator.
Activation of CD4 T cells by Raf-independent effectors of Ras
Authors: Czyzyk Jan , Brogdon Jennifer L. , Badou Abdallah , Henegariu Octavian , Preston Hurlburt Paula , Flavell Richard , Bottomly Kim
Published: May 2003

Small GTPase Ras is capable of mediating activation in T lymphocytes by using Raf kinase-dependent signaling pathway. Other effectors of Ras exist, however, suggesting that targets of Ras alternative to Raf may also contribute to T cell functions. Here we demonstrate that RasV12G37mutant that fails to bind Raf, potently increases intracellular calcium concentration and cytokine production in primary antigen-stimulated T cells. From three known effectors which retain the ability to interact with RasV12G37, overexpression of phospholipase C ɛ but not that of RIN1 or Ral guanine nucleotide exchange factors enhanced cytokine and nuclear factor-activated T cell reporter T cell responses. Hence T cell activation can be critically regulated by the Ras effector pathway independent from Raf that can be mimicked by phospholipase C ɛ.
Polo-like kinase (Plk)1 depletion induces apoptosis in cancer cells
Authors: Liu Xiaoqi , Erikson Raymond L.
Published: May 2003

Elevated expression of mammalian polo-like kinase (Plk)1 occurs in many different types of cancers, and Plk1 has been proposed as a novel diagnostic marker for several tumors. We used the recently developed vector-based small interfering RNA technique to specifically deplete Plk1 in cancer cells. We found that Plk1 depletion dramatically inhibited cell proliferation, decreased viability, and resulted in cell-cycle arrest with 4
N
DNA content. The formation of dumbbell-like chromatin structure suggests the inability of these cells to completely separate the sister chromatids at the onset of anaphase. Plk1 depletion induced apoptosis, as indicated by the appearance of subgenomic DNA in fluorescence-activated cell-sorter (FACS) profiles, the activation of caspase 3, and the formation of fragmented nuclei. Plk1-depletion-induced apoptosis was partially reversed by cotransfection of nondegradable mouse Plk1 constructs. In addition, the p53 pathway was shown to be involved in Plk1-depletion-induced apoptosis. DNA damage occurred in Plk1-depleted cells and inhibition of ATM strongly potentiated the lethality of Plk1 depletion. Although p53 is stabilized in Plk1-depleted cells, DNA damage also occurs in p53
−/−
cells. These data support the notion that disruption of Plk1 function could be an important application in cancer therapy.
DNA relaxation by human topoisomerase I occurs in the closed clamp conformation of the protein
Authors: Carey James F. , Schultz Sharon J. , Sisson Lisa , Fazzio Thomas G. , Champoux James J.
Published: May 2003

In cocrystal structures of human topoisomerase I and DNA, the enzyme is tightly clamped around the DNA helix. After cleavage and covalent attachment of the enzyme to the 3′ end at the nick, DNA relaxation requires rotation of the DNA helix downstream of the cleavage site. Models based on the cocrystal structure reveal that there is insufficient space in the protein for such DNA rotation without some deformation of the cap and linker regions of the enzyme. Alternatively, it is conceivable that the protein clamp opens to facilitate the rotation process. To distinguish between these two possibilities, we engineered two cysteines into the opposing loops of the “lips” region of the enzyme, which allowed us to lock the protein via a disulfide crosslink in the closed conformation around the DNA. Importantly, the rate of DNA relaxation when the enzyme was locked on the DNA was comparable to that observed in the absence of the disulfide crosslink. These results indicate that DNA relaxation likely proceeds without extensive opening of the enzyme clamp.
Membrane receptor trafficking: Evidence of proximal and distal zones conferred by two independent endoplasmic reticulum localization signals
Authors: Shikano Sojin , Li Min
Published: May 2003

The generic membrane trafficking signals of internal RXR and carboxyl-terminal KKXX motifs direct intracellular endoplasmic reticulum (ER) localization of the signal-bearing proteins. These signaling motifs play a critical role in partitioning proteins into designated subcellular compartments by functioning as an intracellular “zip code.” In the process of determining the potential distinctions between these two otherwise functionally identical motifs, two functional zones of these signals were revealed. The KKXX signal was effective only when it was positioned closer to the membrane surface. In contrast, under identical conditions, the internal RXR signal was functional when it was positioned distally from the membrane. Different from the C-terminal KKXX signal, the internal RXR motif may be present in multiple copies. The receptor with multivalent RXR motifs displayed similar trafficking behavior to that of the same receptor with one copy of the RXR motif. The distinctive operating ranges from their anchored membrane surface provide experimental evidence for the notion that there are functional zoning layers within which membrane protein signal motifs are active.
Inducible expression of keratinocyte growth factor (KGF) in mice inhibits lung epithelial cell death induced by hyperoxia
Authors: Ray Prabir , Devaux Yvan , Stolz Donna B. , Yarlagadda Manohar , Watkins Simon C. , Lu Yunbiao , Chen Li , Yang Xiao-fang , Ray Anuradha
Published: May 2003

Oxidant-induced injury to the lung is associated with extensive damage to the lung epithelium. Instillation of keratinocyte growth factor (KGF) in the lungs of animals protects animals from oxidant-induced injury but the mechanism of protection is not well understood. An inherent problem in studying KGF function
in vivo
has been that constitutive overexpression of KGF in the lung causes embryonic lethality with extensive pulmonary malformation. Here we report the development of a stringently regulated, tetracycline-inducible, lung-specific transgenic system that allows regulated expression of KGF in the lung without causing developmental abnormalities from leaky KGF expression. By using this system, we show that exposure of KGF-expressing mice to hyperoxia protects the lung epithelium but not the endothelium from cell death in accordance with the selective expression of KGF receptor on epithelial and not on endothelial cells. Investigations of KGF-induced cell survival pathways revealed KGF-induced activation of the multifunctional pro-survival Akt signaling axis both
in vitro
and
in vivo
. Inhibition of KGF-induced Akt activation by a dominant-negative mutant of Akt blocked the KGF-mediated protection of epithelial cells exposed to hyperoxia. KGF-induced Akt activation may play an important role in inhibiting lung alveolar cell death thereby preserving the lung architecture and function during oxidative stress.
Controlling integration specificity of a yeast retrotransposon
Authors: Zhu Yunxia , Dai Junbiao , Fuerst Peter G. , Voytas Daniel F.
Published: May 2003

Retrotransposons and retroviruses integrate nonrandomly into eukaryotic genomes. For the yeast retrotransposon Ty5, integration preferentially occurs within domains of heterochromatin. Targeting to these locations is determined by interactions between an amino acid sequence motif at the C terminus of Ty5 integrase (IN) called the targeting domain, and the heterochromatin protein Sir4p. Here we show that new Ty5 integration hot spots are created when Sir4p is tethered to ectopic DNA sites. Targeting to sites of tethered Sir4p is abrogated by single amino acid substitutions in either IN or Sir4p that prevent their interaction. Ty5 target specificity can be altered by replacing the IN-targeting domain with other peptide motifs that interact with known protein partners. Integration occurs at high efficiency and in close proximity to DNA sites where the protein partners are tethered. These findings define a mechanism by which retrotransposons shape their host genomes and suggest ways in which retroviral integration can be controlled.
Anthrax lethal factor represses glucocorticoid and progesterone receptor activity
Authors: Webster Jeanette I. , Tonelli Leonardo H. , Moayeri Mahtab , Simons S. Stoney , Leppla Stephen H. , Sternberg Esther M.
Published: May 2003

We report here that a bacterial toxin, anthrax lethal toxin (LeTx), at very low concentrations represses glucocorticoid receptor (GR) transactivation in a transient transfection system and the activity of an endogenous GR-regulated gene in both a cellular system and an animal model. This repression is noncompetitive and does not affect ligand binding or DNA binding, suggesting that anthrax lethal toxin (LeTx) probably exerts its effects through a cofactor(s) involved in the interaction between GR and the basal transcription machinery. LeTx-nuclear receptor repression is selective, repressing GR, progesterone receptor B (PR-B), and estrogen receptor α (ERα), but not the mineralocorticoid receptor (MR) or ERβ. GR repression was also caused by selected p38 mitogen-activated protein (MAP) kinase inhibitors, suggesting that the LeTx action may result in part from its known inactivation of MAP kinases. Simultaneous loss of GR and other nuclear receptor activities could render an animal more susceptible to lethal or toxic effects of anthrax infection by removing the normally protective antiinflammatory effects of these hormones, similar to the increased mortality seen in animals exposed to both GR antagonists and infectious agents or bacterial products. These finding have implications for development of new treatments and prevention of the toxic effects of anthrax.
Generation of neural crest-derived peripheral neurons and floor plate cells from mouse and primate embryonic stem cells
Authors: Mizuseki Kenji , Sakamoto Tatsunori , Watanabe Kiichi , Muguruma Keiko , Ikeya Makoto , Nishiyama Ayaka , Arakawa Akiko , Suemori Hirofumi , Nakatsuji Norio , Kawasaki Hiroshi , Murakami Fujio , Sasai Yoshiki
Published: May 2003

To understand the range of competence of embryonic stem (ES) cell-derived neural precursors, we have examined
in vitro
differentiation of mouse and primate ES cells into the dorsal- (neural crest) and ventralmost (floor plate) cells of the neural axis. Stromal cell-derived inducing activity (SDIA; accumulated on PA6 stromal cells) induces cocultured ES cells to differentiate into rostral CNS tissues containing both ventral and dorsal cells. Although early exposure of SDIA-treated ES cells to bone morphogenetic protein (BMP)4 suppresses neural differentiation and promotes epidermogenesis, late BMP4 exposure after the fourth day of coculture causes differentiation of neural crest cells and dorsalmost CNS cells, with autonomic system and sensory lineages induced preferentially by high and low BMP4 concentrations, respectively. In contrast, Sonic hedgehog (Shh) suppresses differentiation of neural crest lineages and promotes that of ventral CNS tissues such as motor neurons. Notably, high concentrations of Shh efficiently promote differentiation of HNF3β
+
floor plate cells with axonal guidance activities. Thus, SDIA-treated ES cells generate naïve precursors that have the competence of differentiating into the “full” dorsal–ventral range of neuroectodermal derivatives in response to patterning signals.
On the role of MHC class II molecules in the survival and lymphopenia-induced proliferation of peripheral CD4+T cells
Authors: Martin Bruno , Bourgeois Christine , Dautigny Nicole , Lucas Bruno
Published: May 2003

CD4+T cells expand after transfer into lymphopenic H-2bAβ−/−mice (I-Aβ-, I-Eα-deficient mice) but not after transfer into lymphopenic MHC IIΔ/Δmice (I-Aα-, I-Aβ-, I-Eα-, and I-Eβ-deficient mice), implying that in H-2bAβ−/−mice, Aαchain and Eβchain associate to form a hybrid AαEβMHC class II molecule. In light of this unexpected result, we reexamined the MHC class II requirement in the survival and lymphopenia-induced proliferation of CD4+T cells. Here we show that expansion, but not short-term survival, of CD4+T cells depends on interactions with MHC class II molecules in lymphopenic mice. Nevertheless, interactions with classical MHC class II molecules are required for CD4+T cells to survive in CD8+T-cell-containing mice.
Deletion of neuropeptide Y (NPY) 2 receptor in mice results in blockage of NPY-induced angiogenesis and delayed wound healing
Authors: Ekstrand A. Jonas , Cao Renhai , Björndahl Meit , Nyström Susanne , Jönsson-Rylander Ann-Cathrine , Hassani Hessameh , Hallberg Bengt , Nordlander Margareta , Cao Yihai
Published: May 2003

Neuropeptide Y (NPY), a 36-aa peptide, is widely distributed in the brain and peripheral tissues. Whereas physiological roles of NPY as a hormone/neurotransmitter have been well studied, little is known about its other peripheral functions. Here, we report that NPY acts as a potent angiogenic factor
in vivo
using the mouse corneal micropocket and the chick chorioallantoic membrane (CAM) assays. Unlike vascular endothelial growth factor (VEGF), microvessels induced by NPY had distinct vascular tree-like structures showing vasodilation. This angiogenic pattern was similar to that induced by fibroblast growth factor-2, and the angiogenic response was dose-dependent. In the developing chick embryo, NPY stimulated vascular sprouting from preexisting blood vessels. When [Leu
31
Pro
34
]NPY, a NPY-based analogue lacking high affinity for the NPY Y
2
receptor but capable of stimulating both Y
1
and Y
5
receptors, was used in the corneal model, no angiogenic response could be detected. In addition, NPY failed to induce angiogenesis in Y
2
receptor-null mice, suggesting that this NPY receptor subtype was mediating the angiogenic signal. In support of this finding, the Y
2
receptor, but not Y
1
, Y
4
, or Y
5
receptors, was found to be widely expressed in newly formed blood vessels. Further, a delay of skin wound healing with reduced neovascularization was found in Y
2
receptor-null mice. These data demonstrate that NPY may play an important role in the regulation of angiogenesis and angiogenesis-dependent tissue repair.
A cell-based assay for aggregation inhibitors as therapeutics of polyglutamine-repeat disease and validation in
Drosophila
Authors: Apostol Barbara L. , Kazantsev Alexsey , Raffioni Simona , Illes Katalin , Pallos Judit , Bodai Laszlo , Slepko Natalia , Bear James E. , Gertler Frank B. , Hersch Steven , Housman David E. , Marsh J. Lawrence , Thompson Leslie Michels
Published: May 2003

The formation of polyglutamine-containing aggregates and inclusions are hallmarks of pathogenesis in Huntington's disease that can be recapitulated in model systems. Although the contribution of inclusions to pathogenesis is unclear, cell-based assays can be used to screen for chemical compounds that affect aggregation and may provide therapeutic benefit. We have developed inducible PC12 cell-culture models to screen for loss of visible aggregates. To test the validity of this approach, compounds that inhibit aggregation in the PC12 cell-based screen were tested in a
Drosophila
model of polyglutamine-repeat disease. The disruption of aggregation in PC12 cells strongly correlates with suppression of neuronal degeneration in
Drosophila
. Thus, the engineered PC12 cells coupled with the
Drosophila
model provide a rapid and effective method to screen and validate compounds.
Human–Mouse Gene Identification by Comparative Evidence Integration and Evolutionary Analysis
Authors: Zhang Lingang , Pavlovic Vladimir , Cantor Charles R , Kasif Simon
Published: May 2003

The identification of genes in the human genome remains a challenge, as the actual predictions appear to disagree tremendously and vary dramatically on the basis of the specific gene-finding methodology used. Because the pattern of conservation in coding regions is expected to be different from intronic or intergenic regions, a comparative computational analysis can lead, in principle, to an improved computational identification of genes in the human genome by using a reference, such as mouse genome. However, this comparative methodology critically depends on three important factors: (1) the selection of the most appropriate reference genome. In particular, it is not clear whether the mouse is at the correct evolutionary distance from the human to provide sufficiently distinctive conservation levels in different genomic regions, (2) the selection of comparative features that provide the most benefit to gene recognition, and (3) the selection of evidence integration architecture that effectively interprets the comparative features. We address the first question by a novel evolutionary analysis that allows us to explicitly correlate the performance of the gene recognition system with the evolutionary distance (time) between the two genomes. Our simulation results indicate that there is a wide range of reference genomes at different evolutionary time points that appear to deliver reasonable comparative prediction of human genes. In particular, the evolutionary time between human and mouse generally falls in the region of good performance; however, better accuracy might be achieved with a reference genome further than mouse. To address the second question, we propose several natural comparative measures of conservation for identifying exons and exon boundaries. Finally, we experiment with Bayesian networks for the integration of comparative and compositional evidence.
Assessment of SAGE in Transcript Identification
Authors: Pleasance Erin D. , Marra Marco A. , Jones Steven J.M.
Published: May 2003

An essential step in Serial Analysis of Gene Expression (SAGE) is tag mapping, which refers to the unambiguous determination of the gene represented by a SAGE tag. Current resources for tag mapping are incomplete, and thus do not allow assessment of the efficacy of SAGE in transcript identification. A method of tag mapping is described here and applied to the Drosophila melanogaster and Caenorhabditis elegans genomes, which permits detailed SAGE assessment and provides tag-mapping resources that were unavailable previously for these organisms. In our method, a conceptual transcriptome is constructed using genomic sequence and annotation by extending predicted coding regions to include UTRs on the basis of EST and cDNA alignments, UTR length distributions, and polyadenylation signals. Analysis of extracted tags suggests that, using the standard SAGE procedure, expression of 8% of D. melanogaster and 15% of C. elegans
genes cannot be detected unambiguously by SAGE due to shared sequence or lack of NlaIII-anchoring enzyme sites. Both increasing tag length by 2–3 bp and using Sau3A instead of NlaIII as the anchoring enzyme increases potential for transcript detection. This work identifies and quantifies genes not amenable to SAGE analysis, in addition to providing tag-to-gene mappings for two model organisms.