bluuebunny/crossref_metadata_2025 huggingface dataset:
Diadenosine pentaphosphate vasodilates the forearm vascular bed: Inhibition by theophylline and augmentation by dipyridamole
Authors: van Ginneken Egidia E. M. , Rongen Gerard A. , Russel Frans G. M. , Smits Paul
Published: October 2002

Background

In rats, diadenosine pentaphosphate (AP5A) has been implicated in the pathogenesis of essential hypertension. This study describes for the first time the vasomotor action of AP5A in humans by means of the “perfused forearm technique.”

Results

AP5A evoked a dose‐dependent forearm vasodilator response equal to that of adenosine but less than that of adenosine triphosphate (ATP) at equimolar doses. The P1‐purinoceptor antagonist theophylline (0.28 μmol/min per deciliter) reduced the percentage decrease in forearm vascular resistance (FVR) to AP5A (0.6, 6, and 20 nmol/min/dL): −8% ± 6%, −50% ± 6%, and −68% ± 4% during saline solution versus −7% ± 4%, −33% ± 5%, and −45% ± 6% during theophylline (mean ± standard error [SE]; ANOVA for repeated measures; P < .05 for the interaction between purine dose and theophylline; n = 10). An inhibitor of equilibrative nucleoside transport, dipyridamole (7.4 nmol/min per deciliter), augmented the AP5A (0.6 and 6 nmol/min per deciliter)‐induced reduction in FVR as follows: −34% ± 6% and −67% ± 5% during saline versus −49% ± 5% and −80% ± 3% during dipyridamole (P < .05 for the effect of dipyridamole; n = 6). The bivalent cation chelator ethylenediaminetetra‐acetic acid (EDTA) inhibited the rapid degradation of AP5A in vitro. In vivo, the highest tolerated intra‐arterial EDTA dose was not sufficient to inhibit AP5A metabolism.

Conclusion

Intra‐arterial AP5A caused a dose‐dependent reduction in FVR. This is, at least in part, mediated by its degradation product adenosine. The data do not support an in vivo vasoconstrictor action of AP5A, and as such AP5A does not seem likely to contribute to the pathogenesis of primary hypertension in humans.

Clinical Pharmacology & Therapeutics (2002) 71, 448–456; doi: 10.1067/mcp.2002.124469
Pharmacokinetics of plasma enfuvirtide after subcutaneous administration to patientswith human immunodeficiency virus: Inverse Gaussian density absorption and 2‐compartment disposition*
Authors: Zhang Xiaoping , Nieforth Keith , Lang Jean‐Marie , Rouzier‐Panis Regine , Reynes Jacques , Dorr Albert , Kolis Stanley , Stiles Mark R. , Kinchelow Tosca , Patel Indravadan H.
Published: October 2002

Objective

Enfuvirtide (T‐20) is the first of a novel class of human immunodeficiency virus (HIV) drugs that block gp41‐mediated viral fusion to host cells. The objectives of this study were to develop a structural pharmacokinetic model that would adequately characterize the absorption and disposition of enfuvirtide pharmacokinetics after both intravenous and subcutaneous administration and to evaluate the dose proportionality of enfuvirtide pharmacokinetic parameters at a subcutaneous dose higher than that currently used in phase III studies.

Methods

Twelve patients with HIV infection received 4 single doses of enfuvirtide separated by a 1‐week washout period in an open‐label, randomized, 4‐way crossover fashion. The doses studied were 90 mg (intravenous) and 45 mg, 90 mg, and 180 mg (subcutaneous). Serial blood samples were collected up to 48 hours after each dose. Plasma enfuvirtide concentrations were measured with use of a validated liquid chromatography‐tandem mass spectrometry method.

Results

Enfuvirtide plasma concentration‐time data after subcutaneous administration were well described by an inverse Gaussian density function‐input model linked to a 2‐compartment open distribution model with first‐order elimination from the central compartment. The model‐derived mean pharmacokinetic parameters (±SD) were volume of distribution of the central compartment (3.8 ± 0.8 L), volume of distribution of the peripheral compartment (1.7 ± 0.6 L), total clearance (1.44 ± 0.30 L/h), intercompartmental distribution (2.3 ± 1.1 L/h), bioavailability (89% ± 11%), and mean absorption time (7.26 hours, 8.65 hours, and 9.79 hours for the45‐mg, 90‐mg, and 180‐mg dose groups, respectively). The terminal half‐life increased from 3.46 to 4.35hours for the subcutaneous dose range from 45 to 180 mg.

Conclusions

An inverse Gaussian density function‐input model linked to a 2‐compartment open distribution model with first‐order elimination from the central compartment was appropriate to describe complex absorption and disposition kinetics of enfuvirtide plasma concentration‐time data after subcutaneous administration to patients with HIV infection. Enfuvirtide was nearly completely absorbed from subcutaneous depot, and pharmacokinetic parameters were linear up to a dose of 180 mg in this study.

Clinical Pharmacology & Therapeutics (2002) 72, 10–19; doi: 10.1067/mcp.2002.125945
Low‐dose ritonavir moderately enhances nelfinavir exposure
Authors: Kurowski Michael , Kaeser Benoite , Sawyer Anthony , Popescu Matei , Mrozikiewicz Alexander
Published: October 2002

Background

The protease inhibitor ritonavir is increasingly administered at subtherapeutic doses in highly active antiretroviral treatment, to utilize its potential for drug interactions and to enhance the plasma concentrations of other concomitantly prescribed protease inhibitors. The addition of low doses of ritonavir to nelfinavirwas investigated to describe the extent of pharmacokinetic interaction.

Methods

In this randomized, open‐label, one‐sequence crossover study, nelfinavir 1250 mg twice a day was dosed for 17 days, followed by 14 days of nelfinavir 1250 mg twice a day plus low doses of ritonavir of either 100 mg or 200 mg orally. Twenty‐four healthy volunteers were evaluated for pharmacokinetics of nelfinavir, its metabolite M8, andritonavir. Plasma concentrations were measured up to 12 hours after morning and evening dosing, respectively, on days 14 and 31.

Results

Ritonavir increased the area under the plasma concentration‐time curve(AUC) of nelfinavir by 20% (P =.024) and 39% (P =.001) after morning and evening administration, respectively. The AUC of nelfinavir metabolite M8was increased by 74% and 86% after morning and evening dosing (P <.001 for both).

Conclusion

During ritonavir combination therapy a clear although minor drug effect on nelfinavir pharmacokinetics was demonstrated but no dose effect was shown.

Clinical Pharmacology & Therapeutics (2002) 72, 123–132; doi: 10.1067/mcp.2002.126178
Comparison of rofecoxib, celecoxib, and naproxen on renal function in elderly subjects receiving a normal‐salt diet
Authors: Schwartz J. I. , Vandormael K. , Malice M. P. , Kalyani R. N. , Lasseter K. C. , Holmes G. B. , Gertz B. J. , Gottesdiener K. M. , Laurenzi M. , Redfern K.‐J. , Brune K.
Published: October 2002

Background

This study compared directly the renal effects of two selective cyclooxygenase (COX)‐2 inhibitors (rofecoxib and celecoxib) with naproxen (dual COX‐1/COX‐2 inhibitor) and placebo in healthy elderly subjects on a sodium‐replete diet.

Methods

A total of 67 elderly subjects stabilized in the clinic for weight andurinary sodium on a controlled 200‐mEq sodium diet were randomized in a double‐blind fashion to receive rofecoxib, 25 mg daily (n = 17); celecoxib, 200 mg twice daily (n = 17); naproxen, 500 mg twice daily (n = 17); or matching placebo (n = 16) for 28 days. Subjects were sequestered in the clinic for the first 14 treatment days on the controlled diet.

Results

Daily urinary sodium excretion during the first 72 hours of treatment (primary endpoint) significantly decreased in rofecoxib, celecoxib, and naproxen groups compared with baseline (P ≤ .05). Rofecoxib and celecoxib decreases in urinarysodium excretion rates that were comparable with each other, on the basis of predefined boundaries (−39.5 versus −27.1 mEq/d, respectively) and to naproxen (−40.6, mEq/d). Rofecoxib, celecoxib, and naproxen increased mean systolic blood pressure to a similar degree (3.4, 4.3, and 3.1 mm Hg, respectively, versus −1.3 mm Hg for placebo) after 14 days of treatment; small changes also occurred in diastolic blood pressure (0.3, 0.8, and −0.4 mm Hg, respectively, versus −1.4 mm Hg for placebo). Changes from baseline in creatinine clearance, body weight, and urinary potassium excretion among active treatments were similar. After 28 days of treatment, findings were generally consistent with those at 14 days. No subject reported edema or discontinued treatment as the result of an adverse experience.

Conclusion

In healthy elderly subjects on a sodium‐replete diet, the COX‐2 inhibitors rofecoxib and celecoxib did not differ from a nonselective nonsteroidal anti‐inflammatory drug (naproxen), in influencing renal function as measured by urinary sodium excretion, systolic and diastolic blood pressure, creatinine clearance, or weight change.

Clinical Pharmacology & Therapeutics (2002) 72, 50–61; doi: 10.1067/mcp.2002.126182
Severe 2,3,7,8‐tetrachlorodibenzo‐p‐dioxin (TCDD) intoxication: Insights into the measurement of hepatic cytochrome P450 1A2 induction*
Authors: Abraham Klaus , Geusau Alexandra , Tosun Yalcin , Helge Hans , Bauer Steffen , Brockmöller Jürgen
Published: October 2002

Objective

The correct in vivo quantification of aryl hydrocarbon receptor‐mediated induction of cytochrome P450 1A2 (CYP1A2) in humans is a long‐standing question. We compared the performance of several modifications of the caffeine test for measurement of CYP1A2 activity in subjects with exceptionally high, low, or absent enzyme induction.

Methods

CYP1A2 activity was measured in 2 women highly exposed to 2,3,7,8‐tetrachlorodibenzo‐p‐dioxin (TCDD), in 1 man moderately exposed, and in 50 control subjects (30 nonsmokers and 20 heavy smokers). After the application of a test dose, caffeine demethylation was detected with the carbon 13 breath test, the total clearance, and several serum and urinary metabolite ratios.

Results

In the highly TCDD‐exposed persons, results of the breath test (cumulative 15‐minute dose), the total caffeine clearance, the serum metabolic ratio paraxanthine/caffeine (30 and 120 minutes after application), and the urinary metabolic ratio sum of5‐acetylamino‐6‐formylamino‐3‐methyluracil (AFMU), 1‐methyluric acid (1U), and 1‐methylxanthine (1X) over 1,7‐dimethyluric acid (17U) showed a CYP1A2 activity 8 to 10 times higher than the mean of nonsmokers. In contrast, two caffeine urinary metabolic ratios with the parent substance in the denominator did not reflect the CYP1A2 enzyme induction. These ratios strongly depended on urine flow. For the breath test, only results evaluated for ashort sampling period (eg, 15 minutes after application) revealed the high induction. Compared with nonsmokers, higher mean values (maximally 1.8 times) were observed in smokers with all tests.

Conclusion

After high TCDD exposure, hepatic CYP1A2 activity is inducible at least 10 times in humans. Moderate TCDD exposure (up to 1000 ppt in blood fat) does not cause a CYP1A2 induction that can be measured to differentiate from background exposure individually. Therefore direct quantification of such toxins is more specific and sensitive.

Clinical Pharmacology & Therapeutics (2002) 72, 163–174; doi: 10.1067/mcp.2002.126408
Hypoxic pulmonary artery fibroblasts trigger proliferation of vascular smooth muscle cells‐role of hypoxia‐inducible transcription factors
Authors: Rose Frank , Grimminger Friedrich , Appel Jutta , Heller Mathias , Pies Volker , Weissmann Norbert , Fink Ludger , Schmidt Sebastian , Krick Stefanie , Camenisch Gieri , Gassmann Max , Seeger Werner , Hänze Jörg
Published: October 2002

Chronic lung hypoxia causes vascular remodeling with pulmonary artery smooth muscle cell (SMCPA) hyperplasia, resulting in pulmonary hypertension and cor pulmonale. We investigated SMCPA and pulmonary artery adventitial fibroblasts (FBPA) for their proliferative response to hypoxia. Strong SMCPA growth occurred under hypoxic conditions in SMCPA/FBPA co‐cultures, but not in SMCPA monocultures. SMCPA growth was fully reproduced by transferring serum‐free supernatant from hypoxic cultured FBPA to normoxic SMCPA. Hypoxia‐inducible‐transcriptionfactor subtypes (HIF‐1α, HIF‐2α, HIF‐3α) and its dependent target genes, carrying the hypoxiaresponsive‐element as regulatory component, were strongly activated in both hypoxic FBPA and SMCPA. HIF‐transcription‐factor decoy technique, employed to FBPA during hypoxic culturing, blocked the mitogenic activity of FBPA conditioned medium on SMCPA. The data suggest that hypoxia‐driven gene regulation in pulmonary artery fibroblasts results in a mitogenic stimulus on adjacent pulmonary artery smooth muscle cells, and HIF‐transcription‐decoy may offer a new therapeutic approach to suppress these events.
Extracting 3D from Motion: Differences in Human and Monkey Intraparietal Cortex
Authors: Vanduffel W. , Fize D. , Peuskens H. , Denys K. , Sunaert S. , Todd J. T. , Orban G. A.
Published: October 2002

We compared three-dimensional structure-from-motion (3D-SFM) processing in awake monkeys and humans using functional magnetic resonance imaging. Occipital and midlevel extrastriate visual areas showed similar activation by 3D-SFM stimuli in both species. In contrast, intraparietal areas showed significant 3D-SFM activation in humans but not in monkeys. This suggests that human intraparietal cortex contains visuospatial processing areas that are not present in monkeys.
Molecular Characterization of the Growth Phase-Dependent Expression of the
lsrA
Gene, Encoding Levansucrase of
Rahnella aquatilis
Authors: Seo Jeong-Woo , Jang Ki-Hyo , Kang Soon Ah , Song Ki-Bang , Jang Eun Kyung , Park Buem-Seek , Kim Chul Ho , Rhee Sang-Ki
Published: October 2002

Expression of the
lsrA
gene from
Rahnella aquatilis
, encoding levansucrase, is tightly regulated by the growth phase of the host cell; low-level expression was observed in the early phase of cell growth, but expression was significantly stimulated in the late phase. Northern blot analysis revealed that regulation occurred at the level of transcription. The promoter region was identified by primer extension analysis. Two opposite genetic elements that participate in the regulation of
lsrA
expression were identified upstream of the
lsrA
gene: the
lsrS
gene and the
lsrR
region. The
lsrS
gene encodes a protein consisting of 70 amino acid residues (
M
r
, 8,075), which positively activated
lsrA
expression approximately 20-fold in a growth phase-dependent fashion. The
cis
-acting
lsrR
region, which repressed
lsrA
expression about 10-fold, was further narrowed to two DNA regions by deletion analysis. The concerted action of two opposite regulatory functions resulted in the growth phase-dependent activation of gene expression in
Escherichia coli
independent of the stationary sigma factor σ
S
.
Converting the NiFeS Carbon Monoxide Dehydrogenase to a Hydrogenase and a Hydroxylamine Reductase
Authors: Heo Jongyun , Wolfe Marcus T. , Staples Christopher R. , Ludden Paul W.
Published: October 2002

Substitution of one amino acid for another at the active site of an enzyme usually diminishes or eliminates the activity of the enzyme. In some cases, however, the specificity of the enzyme is changed. In this study, we report that the changing of a metal ligand at the active site of the NiFeS-containing carbon monoxide dehydrogenase (CODH) converts the enzyme to a hydrogenase or a hydroxylamine reductase. CODH with alanine substituted for Cys531exhibits substantial uptake hydrogenase activity, and this activity is enhanced by treatment with CO. CODH with valine substituted for His265exhibits hydroxylamine reductase activity. Both Cys531and His265are ligands to the active-site cluster of CODH. Further, CODH with Fe substituted for Ni at the active site acquires hydroxylamine reductase activity.
DNA Inversion on Conjugative Plasmid pVT745
Authors: Chen Jinbiao , Leblanc Donald J. , Galli Dominique M.
Published: October 2002

Plasmid pVT745 from
Actinobacillus actinomycetemcomitans
strain VT745 can be transferred to other
A. actinomycetemcomitans
strains at a frequency of 10
−6
. Screening of transconjugants revealed that the DNA of pDMG21A, a pVT745 derivative containing a kanamycin resistance gene, displayed a structural rearrangement after transfer. A 9-kb segment on the plasmid had switched orientation. The inversion was independent of RecA and required the activity of the pVT745-encoded site-specific recombinase. This recombinase, termed Inv, was highly homologous to invertases of the Din family. Two recombination sites of 22 bp, which are arranged in opposite orientation and which function as DNA crossover sequences, were identified on pVT745. One of the sites was located adjacent to the 5′ end of the invertase gene,
inv
. Inversion of the 9-kb segment on pVT745 derivatives has been observed in all
A. actinomycetemcomitans
strains tested except for the original host, VT745. This would suggest that a host factor that is either inactive or absent in VT745 is required for efficient recombination. Inactivation of the invertase in the donor strain resulted in a 1,000-fold increase in the number of transconjugants upon plasmid transfer. It is proposed that an activated invertase causes the immediate loss of the plasmid in most recipient cells after mating. No biological role has been associated with the invertase as of yet.
The First Archaeal ATP-Dependent Glucokinase, from the Hyperthermophilic Crenarchaeon
Aeropyrum pernix
, Represents a Monomeric, Extremely Thermophilic ROK Glucokinase with Broad Hexose Specificity
Authors: Hansen Thomas , Reichstein Bianca , Schmid Roland , Schönheit Peter
Published: October 2002

An ATP-dependent glucokinase of the hyperthermophilic aerobic crenarchaeon
Aeropyrum pernix
was purified 230-fold to homogeneity. The enzyme is a monomeric protein with an apparent molecular mass of about 36 kDa. The apparent

K
m

values for ATP and glucose (at 90°C and pH 6.2) were 0.42 and 0.044 mM, respectively; the apparent
V
max
was about 35 U/mg. The enzyme was specific for ATP as a phosphoryl donor, but showed a broad spectrum for phosphoryl acceptors: in addition to glucose, which showed the highest catalytic efficiency (
k
cat
/

K
m

), the enzyme also phosphorylates glucosamin, fructose, mannose, and 2-deoxyglucose. Divalent cations were required for maximal activity: Mg
2+
, which was most effective, could partially be replaced with Co
2+
, Mn
2+
, and Ni
2+
. The enzyme had a temperature optimum of at least 100°C and showed significant thermostability up to 100°C. The coding function of open reading frame (ORF) APE2091 (Y. Kawarabayasi, Y. Hino, H. Horikawa, S. Yamazaki, Y. Haikawa, K. Jin-no, M. Takahashi, M. Sekine, S. Baba, A. Ankai, H. Kosugi, A. Hosoyama, S. Fukui, Y. Nagai, K. Nishijima, H. Nakazawa, M. Takamiya, S. Masuda, T. Funahashi, T. Tanaka, Y. Kudoh, J. Yamazaki, N. Kushida, A. Oguchi, and H. Kikuchi, DNA Res. 6:83-101, 145-152, 1999), previously annotated as gene
glk
, coding for ATP-glucokinase of
A. pernix
, was proved by functional expression in
Escherichia coli
. The purified recombinant ATP-dependent glucokinase showed a 5-kDa higher molecular mass on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, but almost identical kinetic and thermostability properties in comparison to the native enzyme purified from
A. pernix.
N-terminal amino acid sequence of the native enzyme revealed that the translation start codon is a GTG 171 bp downstream of the annotated start codon of ORF APE2091. The amino acid sequence deduced from the truncated ORF APE2091 revealed sequence similarity to members of the ROK family, which comprise bacterial sugar kinases and transcriptional repressors. This is the first report of the characterization of an ATP-dependent glucokinase from the domain of
Archaea
, which differs from its bacterial counterparts by its monomeric structure and its broad specificity for hexoses.
The Dilemma of Phage Taxonomy Illustrated by Comparative Genomics of Sfi21-LikeSiphoviridaein Lactic Acid Bacteria
Authors: Proux Caroline , van Sinderen Douwe , Suarez Juan , Garcia Pilar , Ladero Victor , Fitzgerald Gerald F. , Desiere Frank , Brüssow Harald
Published: October 2002

The complete genome sequences of two dairy phages,Streptococcus thermophilusphage 7201 andLactobacillus caseiphage A2, are reported. Comparative genomics reveals that both phages are members of the recently proposed Sfi21-like genus ofSiphoviridae, a widely distributed phage type in low-GC-content gram-positive bacteria. Graded relatedness, the hallmark of evolving biological systems, was observed when different Sfi21-like phages were compared. Across the structural module, the graded relatedness was represented by a high level of DNA sequence similarity or protein sequence similarity, or a shared gene map in the absence of sequence relatedness. This varying range of relatedness was found within Sfi21-like phages from a single species as demonstrated by the different prophages harbored byLactococcus lactisstrain IL1403. A systematic dot plot analysis with 11 completeL. lactisphage genome sequences revealed a clear separation of all temperate phages from two classes of virulent phages. The temperate lactococcal phages share DNA sequence homology in a patchwise fashion over the nonstructural gene cluster. With respect to structural genes, four DNA homology groups could be defined within temperateL. lactisphages. Closely related structural modules for all four DNA homology groups were detected in phages fromStreptococcusorListeria, suggesting that they represent distinct evolutionary lineages that have not uniquely evolved inL. lactis. It seems reasonable to base phage taxonomy on data from comparative genomics. However, the peculiar modular nature of phage evolution creates ambiguities in the definition of phage taxa by comparative genomics. For example, depending on the module on which the classification is based, temperate lactococcal phages can be classified as a single phage species, as four distinct phage species, or as two if not three different phage genera. We propose to base phage taxonomy on comparative genomics of a single structural gene module (head or tail genes). This partially phylogeny-based taxonomical system still mirrors some aspects of the current International Committee on Taxonomy in Virology classification system. In this system the currently sequenced lactococcal phages would be grouped into five genera: c2-, sk1, Sfi11-, r1t-, and Sfi21-like phages.
Conserved Filamentous Prophage in
Escherichia coli
O18:K1:H7 and
Yersinia pestis
Biovar orientalis
Authors: Gonzalez Mark D. , Lichtensteiger Carol A. , Caughlan Ruth , Vimr Eric R.
Published: October 2002

Microbial virulence is known to emerge by horizontal gene transfer mechanisms. Here we describe the discovery of a novel filamentous prophage, designated CUS-1, which is integrated into the chromosomal
dif
homologue of the high-virulence clone
Escherichia coli
O18:K1:H7. An homologous chromosomal element (CUS-2) in
Yersinia pestis
biovar orientalis is integrated at the same relative location as CUS-1; both lysogenic
E. coli
and
Y. pestis
strains produce particles with properties expected of single-stranded DNA virions. CUSφ is epidemiologically correlated with the emergence of K1 strains with increased virulence and with the
Y. pestis
biovar responsible for the current (third) plague pandemic.
Effects of a Nod-factor-overproducing strain of Sinorhizobium meliloti on the expression of the ENOD40 gene in Melilotus alba
Authors: Giordano Walter F , Lum Michelle R , Hirsch Ann M
Published: October 2002

We have initiated studies on the molecular biology and genetics of white sweetclover (Melilotus alba Desr.) and its responses to inoculation with the nitrogen-fixing symbiont Sinorhizobium meliloti. Early nodulin genes such as ENOD40 serve as markers for the transition from root to nodule development even before visible stages of nodule formation are evident. Using Northern blot analysis, we found that the ENOD40 gene was expressed within 6 h after inoculation with two different strains of S. meliloti, one of which overproduces symbiotic Nod factors. Inoculation with this strain resulted in an additional increase in ENOD40 gene expression over a typical wild-type S. meliloti strain. Moreover, the increase in mRNA brought about by the Nod-factor-overproducing strain 24 h after inoculation was correlated with lateral root formation by using whole-mount in situ hybridization to localize ENOD40 transcripts in lateral root meristems and by counting lateral root initiation sites. Cortical cell divisions were not detected. We also found that nodulation occurred more rapidly on white sweetclover in response to the Nod-factor-overproducing strain, but ultimately there was no difference in nodulation efficiency in terms of nodule number or the number of roots nodulated by the two strains. Also, the two strains could effectively co-colonize the host when inoculated together, although a few host cells were occupied by both strains.Key words: ENOD40, Nod factor, Melilotus, Sinorhizobium, symbiosis.
Plant pyruvate-dependent gamma-aminobutyrate transaminase: identification of anArabidopsiscDNA and its expression inEscherichia coli
Authors: Van Cauwenberghe Owen R , Makhmoudova Amina , McLean Michael D , Clark Shawn M , Shelp Barry J
Published: October 2002

Both pyruvate- and 2-oxoglutarate-dependent gamma-aminobutyrate transaminase (GABA-T) activities are present in crude tobacco (Nicotiana tabacum L.) leaf extracts. In this study, GABA:pyruvate-T activity was partially purified using mitochondrial isolation and protein solubilization in 3-[3-cholamidopropyl)dimethylammonio]-1-propanesulfonate, and a combination of chromatographic and electrophoretic procedures. A partial amino acid sequence of the putative 55-kDa GABA-T subunit enabled identification of a predicted Arabidopsis thaliana (L.) Heynh. GABA:pyruvate-T expressed sequence tag and subsequent amplification of a 1515 bp open reading frame encoding a 504-amino acid polypeptide. Computer analysis using web-based tools revealed the presence of a putative mitochondrial signal sequence and a pyridoxal-5-phosphate binding domain in the polypeptide. Functional expression of the GABA-T cDNA in Escherichia coli revealed that the recombinant protein uses pyruvate but not 2-oxoglutarate. The Arabidopsis GABA:pyruvate-T cDNA could form the basis for identification of multiple GABA-T isoforms and generation of GABA-T mutants for determining the fate of GABA nitrogen and elucidating the physiological function of GABA in plants.Key words: amino acceptor, gamma-aminobutyrate, gamma-aminobutyrate transaminase, protein purification, heterologous expression, recombinant protein.
Synthesis of disaccharide congeners of theTrichinella spiralisglycan and binding site mapping of two monoclonal antibodies
Authors: Zhang Ping , Appleton Judith , Ling Chang-Chun , Bundle David R
Published: October 2002

The tetrasaccharide epitope, β-D-Tyvp(1[Formula: see text]3)β-D-GalNAcp(1[Formula: see text]4)[α-L-Fucp(1[Formula: see text]3)]β-D-GlcNAcp (1) is the major constituent of the N-glycan expressed on the cell surface of the parasite Trichinella spiralis. Two monoclonal antibodies (Mabs 9D4 and 18H1) that protect rats against infection by T. spiralis bind the terminal disaccharide epitope β-D-Tyvp(1[Formula: see text]3)β-D-GalNAcp conjugated to BSA. The syntheses of disaccharide congeners containing mono-deoxy, mono-methyl, as well as modifications to replace the acetamido group are reported. These target disaccharides were assayed for binding to the protective MAbs. For each antibody different clusters of three hydroxyl groups, that include C-2 and C-4 of tyvelose and for 18H1, the GalNAc acetamido group, provide the key polar interactions with the antibody binding sites. Mapping of the sites by functional group replacement revealed a similar pattern of recognition for the dideoxyhexose by the two MAbs while each recognizes distinct surfaces of the GalNAc residue. Consequently although both antibodies bury the 4-OH of tyvelose, the principal contact surface occurs on opposite sides of the 3,6-dideoxyhexose.Key words: β-tyveloside, 3,6-dideoxy-D-arabino-hexose, Trichinella carbohydrate antigen, antibody mapping, Trichinella spiralis, N-glycans, molecular recognition of carbohydrates, antigen topology, functional group replacement.
Serological and conformational properties ofE. coliK92 capsular polysaccharide and itsN-propionylated derivative both illustrate that induced antibody does not recognize extended epitopes of polysialic acid: Implications for a comprehensive conjugate vaccine against groups B and CN. meningitidis
Authors: Pon Robert A , Khieu Nam Huan , Yang Qing-Ling , Brisson Jean-Robert , Jennings Harold J
Published: October 2002

The capsular polysaccharide of E. coli K92 (K92P) contains elements in common with the capsular polysaccharides of both groups B and C N. meningitidis, and may therefore form the basis of a bivalent vaccine. In an attempt to augment the cross-protective immune response to group B meningococci, the N-acetyl groups of the K92P were replaced by N-propionyl groups (NPrK92P) and conjugated to protein. This strategy had previously been applied with success to the poorly immunogenic capsular polysaccharide of group B meningococcus (GBMP), and the bactericidal epitope was found to be exclusively mimicked by extended helical segments of the NPrGBMP. The NPrK92P-conjugate, in relation to a K92P-conjugate, failed to enhance the response to GBMP but did generate a measurable response to NPrGBMP, but only at the expense of a greatly reduced GCMP response. Despite the presence of an immune response to NPrGBMP, the anti-NPrK92 serum was not bactericidal. Competitive inhibition studies with NPrGBMP oligosaccharides suggested the NPrK92 antibodies could not cross-react with the protective epitope on group B meningococci, as defined by extended helical segments of the NPrGBMP, but only recognized short non-bactericidal NPrGBMP epitopes. This hypothesis was supported from the conformational and molecular dynamics studies of the K92P, which demonstrated a lack of extended conformations that resemble the GBMP extended epitope. Indeed, the conformational properties of the K92P more closely resembled those of the GCMP, thereby explaining the observed moderate cross-protection of the K92P antiserum towards group C meningococci. Thus, on the basis of these results, it can be concluded that K92P, regardless of N-propionyl modification, will not serve as an effective single vaccine component against both groups B and C meningococci.Key words: conjugate vaccine, Neisseria meningitidis, polysialic acid, NMR, molecular dynamics.
Soil nutrition and temperature as drivers of root suckering in trembling aspen
Authors: Fraser Erin C , Lieffers Victor J , Landhäusser Simon M , Frey Brent R
Published: October 2002

In two separate studies, the suckering of trembling aspen (Populus tremuloides Michx.) roots was assessed in response to different daily maximum soil temperatures and in relation to available nutrients (CaSO4 and NH4NO3). In the first study, aspen root sections were incubated under high:low temperature regimes of 12:8, 14:8, 16:8, 18:8, or 20:8°C until 124 degree-days above 8°C had been reached. Daily maximum temperature did not affect the number of suckers produced per square centimetre of root surface area or per root section. However, more time was required to initiate suckers on root sections grown under the 12:8°C temperature regime compared with those grown at 20:8°C. Furthermore, when calculated from a base temperature of 5°C, the number of degree-days needed to initiate aspen suckers was not different across the temperature regimes. In the second study, CaSO4, NH4NO3, or distilled water was added to aspen root sections. While nutrients did not affect the number of suckers produced, the addition of CaSO4 or NH4NO3 significantly increased sucker dry mass. Nutrient addition appeared to stimulate sucker growth and mobilization of stored reserves to support this growth.
FIELD-THEORETIC FOUNDATION OF NO-FIELD QUANTUM HALL EFFECT
Authors: SUGAHARA MASANORI , BOGOLUBOV NIKOLAI N.
Published: October 2002

Recently, the authors discussed the possibility of the macroscopic quantum state similar to the Quantum Hall Effect in a semi-localized 2D electron system with a toroidal electron-wave amplitude in the absence of any magnetic field. In order to give the concrete statistical foundation of the study, the fermion-boson statistical transformation of the 2D electron system is made using a Chern–Simons gauge potential. Based on the solution of the resultant boson-type Hamiltonian, we construct the fermion-type solution via a unitary transformation. It is shown that the solution in the form of Laughlin function is stable when electrons form pairs. In the presence of hole doping, the pair Laughlin function leads to a representation of a superconducting state when the phase-coherence length λΘ exceeds the incompressibility length λQ, but when λΘ< λQ, it leads to a macroscopic quantum state characterized by particle-number definiteness.
DEPENDENCE OF THE GLASS TRANSITION TEMPERATURE ON DENSITY IN A SOFT SPHERE SYSTEM WITH MORSE POTENTIAL
Authors: LIU HAO-YANG , ZHANG WEN-BING , ZOU XIAN-WU , YUAN YIN-QUAN , JIN ZHUN-ZHI
Published: October 2002

The glass transition for the soft sphere system with Morse potential was investigated based on molecular dynamics simulation and partition function analysis. The dependence of the glass transition temperature on the density and the softness of the potential was obtained.
COMPONENT CONTRACT-BASED INTERFACE SPECIFICATION TECHNIQUE USING Z
Authors: LEE JI-HYUN , YOO CHEOL-JUNG , CHANG OK-BAE
Published: October 2002

A simple contract contains formalizing preconditions, postconditions, and invariants. A contract describes the services that are provided by an object. In the component world, it is essential to describe correctly what services are provided by the components and how we can use them. Till now, interfaces are specified in IDL, which describes the syntactic aspect of interface. IDL cannot describe the semantic aspects and safety condition of a component that a client and a server must know how to use or implement it. In this paper, we use contracts specifying components and propose an interface specification technique to describe components as a contract(namely, component contract). We regard a component's interface as contract and present which aspect to be specified for providing correct information of the components. This paper defines some definitions and describes operators such as cooperating components, component version, functional requirements, nonfunctional requirements, and performance measurements to specify components, and specifies them using Z scheme. Finally, we apply this interface specification technique to develop an e-commerce system.
The G20210A mutation does not affect the stability of prothrombin mRNA in vivo
Authors: Pollak Eleanor S. , Lam Ho-Sun , Russell J. Eric
Published: October 2002

The activated form of prothrombin plays pivotal roles in the regulation of crucial coagulation, fibrinolytic, and cellular processes. Among several congenital genetic defects affecting the prothrombin gene, a G→A mutation at position 20210—the accepted polyadenylation site—has been linked to hyperprothrombinemia and a corresponding increase in venous and arterial thrombotic risk. The current study substantiates the hypothesis that the 20210A mutation effects posttranscriptional dysregulation of the prothrombin messenger RNA (mRNA). Moreover, data from experiments carried out in fresh liver tissue indicate that the 20210A mutation does not affect prothrombin mRNA stability but, rather, effects a change in the location of the 3′-cleavage/polyadenylation reaction. Based upon this evidence, we propose an alternate model for the dysregulated expression of the prothrombin 20210A gene that does not require a change in the stability of its mRNA.
Transient hematologic and clinical effect of E21R in a child with end-stage juvenile myelomonocytic leukemia
Authors: Bernard Frédéric , Thomas Caroline , Emile Jean François , Hercus Timothy , Cassinat Bruno , Chomienne Christine , Donadieu Jean
Published: October 2002

E21R is a modified granulocyte macrophage–colony-stimulating factor (GM-CSF) protein which results in antagonism of GM-CSF function via selective binding to the GM-CSF receptor complex. Juvenile chronic myelomonocytic leukemia (JMML) is a rare leukemia where spontaneous proliferation of myeloid and monocytic precursors in patients' bone marrow cultures is dependent on GM-CSF. For patients who progress after systemic chemotherapy, there are no effective therapies. In vitro and in vivo studies in an animal model demonstrating that E21R exerts an antileukemic action prompted us to consider its potential utility in a child with end-stage JMML. E21R was well-tolerated during the 3 courses of subcutaneous treatment. A clear in vivo efficacy was observed after 2 courses of E21R but the disease appeared completely refractory during the third course. This novel therapeutic approach clearly deserves further evaluation in JMML.
Homozygous factor V splice site mutation associated with severe factor V deficiency
Authors: Schrijver Iris , Koerper Marion A. , Jones Carol D. , Zehnder James L.
Published: October 2002

We investigated a family whose proband has a severe bleeding disorder and factor V antigenic and functional levels of 8% and less than 1% of control values, respectively. Molecular analysis of the factor V gene revealed a novel homozygous mutation in the last nucleotide of exon 10. 1701G>T causes activation of a cryptic exonic splice site in exon 10, which encodes part of the factor V heavy chain (A2 domain). This leads to the deletion of 35 nucleotides and results in a frameshift with a premature stop codon at amino acid position 498. The G1701 and corresponding Gln509 are conserved in murine, bovine, and porcine factor V and in human factor VIII. Few factor V deficiency mutations have been identified as yet. Several are present in the heterozygous form in combination with factor V Leiden (Arg506Gln). This is the first reported homozygous splice site mutation in a patient with factor V deficiency.
High-dose chemo-radioimmunotherapy with autologous stem cell support for relapsed mantle cell lymphoma
Authors: Gopal Ajay K. , Rajendran Joseph G. , Petersdorf Stephen H. , Maloney David G. , Eary Janet F. , Wood Brent L. , Gooley Theodore A. , Bush Sharon A. , Durack Lawrence D. , Martin Paul J. , Matthews Dana C. , Appelbaum Frederick R. , Bernstein Irwin D. , Press Oliver W.
Published: October 2002

Relapsed mantle cell lymphoma is a radiation-sensitive malignancy that is unlikely to be cured by treatment with conventional high-dose therapy and autologous stem cell transplantation. We tested the safety and efficacy of using a CD20-specific monoclonal antibody conjugated with 131I to deliver high-dose radiation selectively to all lymphoma sites. Patients with relapsed or refractory mantle cell lymphoma received infusions of 131I-labeled CD20-specific monoclonal antibody (Tositumomab). The antibody dose was 1.7 mg/kg body weight, and the amount of 131I was calibrated to deliver 20 to 25 Gy to vital normal organs. This treatment was followed 10 days later by administration of high-dose etoposide (30-60 mg/kg), cyclophosphamide (60-100 mg/kg), and infusion of cryopreserved autologous stem cells. The 16 patients in this study had received a median of 3 prior treatments, and 7 had chemotherapy-resistant disease. The median dose of 131I was 510 mCi (18.87 GBq). There were no therapy-related deaths. Among the 11 patients with conventionally measurable disease at the time of treatment, the respective complete and overall response rates were 91% and 100%. Fifteen patients remain alive, and 12 have had no progression of lymphoma at 6 to 57 months from transplantation and 16 to 97 months from diagnosis. Overall survival at 3 years from transplantation is estimated at 93%, and progression-free survival is estimated at 61%. High-dose treatment with 131I-Tositumomab, etoposide, and cyclophosphamide results in a high remission rate and may provide long-term disease-free survival for patients with relapsed or refractory mantle cell lymphoma.
Inhibition of PDE4 phosphodiesterase activity induces growth suppression, apoptosis, glucocorticoid sensitivity, p53, and p21WAF1/CIP1 proteins in human acute lymphoblastic leukemia cells
Authors: Ogawa Ryosuke , Streiff Michael B. , Bugayenko Artem , Kato Gregory J.
Published: October 2002

Glucocorticoids are integral to successful treatment of childhood acute lymphoblastic leukemia (ALL) and other lymphoid malignancies. A large body of data indicates that in various model systems, elevation of cyclic adenosine monophosphate (cAMP) can potentiate glucocorticoid response, although this has not been well evaluated as a potential leukemia treatment. Although cAMP analogs have been studied, little data exist regarding the potential toxicity to leukemia cells of pharmacologic elevation of cAMP levels in leukemic blasts. Using MTT assays of cell proliferation on CEM ALL cells, we found that aminophylline and other nonspecific phosphodiesterase (PDE) inhibitors suppress cell growth. This effect is replicated by the PDE4-specific PDE inhibitor rolipram, but not by specific inhibitors of the PDE1 or PDE3 classes. We found that PDE inhibitors cause increased dexamethasone sensitivity and a synergistic effect with the adenylyl cyclase activator forskolin. We observed several important cellular characteristics associated with this treatment, including elevation of cAMP, induction of p53 and p21WAF1/CIP1proteins, G1 and G2/M cell cycle arrest, and increased apoptosis. Sensitivity to forskolin and rolipram is shared by at least 2 pediatric ALL cell lines, CEM and Reh cells. Some cell lines derived from adult-type lymphoid malignancies also show sensitivity to this treatment. These findings suggest that PDE inhibitors have therapeutic potential in human ALL and characterize the molecular mechanisms that may be involved in this response.
BRCAPRO Validation, Sensitivity of Genetic Testing of BRCA1/BRCA2, and Prevalence of Other Breast Cancer Susceptibility Genes
Authors: Berry Donald A. , Iversen Edwin S. , Gudbjartsson Daniel F. , Hiller Elaine H. , Garber Judy E. , Peshkin Beth N. , Lerman Caryn , Watson Patrice , Lynch Henry T. , Hilsenbeck Susan G. , Rubinstein Wendy S. , Hughes Kevin S. , Parmigiani Giovanni
Published: October 2002

PURPOSE: To compare genetic test results for deleterious mutations of BRCA1 and BRCA2 with estimated probabilities of carrying such mutations; to assess sensitivity of genetic testing; and to assess the relevance of other susceptibility genes in familial breast and ovarian cancer.

PATIENTS AND METHODS: Data analyzed were from six high-risk genetic counseling clinics and concern individuals from families for which at least one member was tested for mutations at BRCA1 and BRCA2. Predictions of genetic predisposition to breast and ovarian cancer for 301 individuals were made using BRCAPRO, a statistical model and software using Mendelian genetics and Bayesian updating. Model predictions were compared with the results of genetic testing.

RESULTS: Among the test individuals, 126 were Ashkenazi Jewish, three were male subjects, 243 had breast cancer, 49 had ovarian cancer, 34 were unaffected, and 139 tested positive for BRCA1 mutations and 29 for BRCA2 mutations. BRCAPRO performed well: for the 150 probands with the smallest BRCAPRO carrier probabilities (average, 29.0%), the proportion testing positive was 32.7%; for the 151 probands with the largest carrier probabilities (average, 95.2%), 78.8% tested positive. Genetic testing sensitivity was estimated to be at least 85%, with false-negatives including mutations of susceptibility genes heretofore unknown.

CONCLUSION: BRCAPRO is an accurate counseling tool for determining the probability of carrying mutations of BRCA1 and BRCA2. Genetic testing for BRCA1 and BRCA2 is highly sensitive, missing an estimated 15% of mutations. In the populations studied, breast cancer susceptibility genes other than BRCA1 and BRCA2 either do not exist, are rare, or are associated with low disease penetrance.
Triplet Combination With Irinotecan Plus Oxaliplatin Plus Continuous-Infusion Fluorouracil and Leucovorin as First-Line Treatment in Metastatic Colorectal Cancer: A Multicenter Phase II Trial
Authors: Souglakos J. , Mavroudis D. , Kakolyris S. , Kourousis Ch. , Vardakis N. , Androulakis N. , Agelaki S. , Kalbakis K. , Tsetis D. , Athanasiadis N. , Samonis G. , Georgoulias V.
Published: October 2002

PURPOSE: To evaluate the efficacy and tolerance of irinotecan (CPT-11) in combination with oxaliplatin (l-OHP) plus fluorouracil (5-FU)/leucovorin (LV) (de Gramont regimen) as first-line treatment of metastatic colorectal cancer (MCC).

PATIENTS AND METHODS: Thirty-one patients with MCC who had not received prior therapy for metastatic disease were enrolled. Their median age was 60 years; performance status (World Health Organization) was 0 in 12, 1 in 14, and 2 in five patients; 19 patients (61%) had prior surgery, and 14 (45%) had adjuvant chemotherapy. CPT-11 was administered on day 1 at 150 mg/m2 as a 90-minute intravenous (IV) infusion; l-OHP was administered on day 2 at 65 mg/m2 as a 2-hour IV infusion; and on days 2 and 3, LV 200 mg/m2 preceded 5-FU administration of 400 mg/m2/d initial IV bolus dose followed by 600 mg/m2/d 22-hour IV continuous infusion. The regimen was repeated every 2 weeks.

RESULTS: All patients were assessable for toxicity and 30 for response to treatment. Complete response was achieved in two patients (6.5%) and partial response in 16 (51.6%) (overall response rate, 58.1%; 95% confidence interval, 40.7% to 75.4%); eight patients (25.8%) had stable disease, and five (16.1%) had disease progression. The median duration of response was 9 months, and the median time to disease progression was 13 months. Neutropenia grade 3 to 4 occurred in 14 patients (45%) and febrile neutropenia in two (6%). Diarrhea grade 3 to 4 was observed in 10 patients (32%), neurotoxicity grade 3 to 4 in three (9%), and asthenia grade 3 in two (10%). No treatment-related death has occurred.

CONCLUSION: The triplet combination of 5-FU/LV + CPT-11 + l-OHP is a highly active regimen with manageable toxicity as front-line treatment in MCC.
Temozolomide Plus Thalidomide in Patients With Advanced Melanoma: Results of a Dose-Finding Trial
Authors: Hwu Wen-Jen , Krown Susan E. , Panageas Katherine S. , Menell Jennifer H. , Chapman Paul B. , Livingston Philip O. , Williams Linda J. , Quinn Carolyn J. , Houghton Alan N.
Published: October 2002

PURPOSE: To establish a safe and tolerated regimen of an oral cytotoxic agent, temozolomide, and a cytostatic agent, thalidomide, in patients with unresectable stage III or IV malignant melanoma.

PATIENTS AND METHODS: Patients with unresectable stage III or IV melanoma without brain metastases were entered successively onto four treatment cohorts: level 1, temozolomide 50 mg/m2/d for 6 weeks followed by a 4-week break; levels 2, 3, and 4, temozolomide 75 mg/m2/d for 6 weeks followed, respectively, by breaks of 4, 3, and 2 weeks. Thalidomide was started at 200 mg/d, and escalated to a maximum dose of 400 mg/d. Safety was assessed at weeks 2 and 4 and every 4 weeks thereafter; tumor response was evaluated every 8 to 10 weeks.

RESULTS: Twelve patients were enrolled, three on each cohort. Therapy was generally well tolerated on all of the treatment schedules. Thalidomide at a dose of 400 mg/d was well tolerated in patients younger than 70, and 200 mg/d was well tolerated in older patients. The most common adverse events were grade 2 or 3 constipation and neuropathy, which were attributed to thalidomide. Five major responses (one complete, four partial) were documented, all at dose levels 2 to 4. Three of the five responding patients were in the over-70 age group. The median duration of response was 6 months (range, 4 to 17+ months), and the median overall survival was 12.3 months (range, 4 to 19+ months).

CONCLUSION: The combination of temozolomide and thalidomide was well tolerated and had antitumor activity in patients with advanced melanoma, including elderly patients over 70 years old.