bluuebunny/crossref_metadata_2025 huggingface dataset:
Endothelin-1 Promotes Ca
2+
Antagonist-Insensitive Coronary Smooth Muscle Contraction Via Activation of ε-Protein Kinase C
Authors: McNair Lashonn L. , Salamanca Daisy A. , Khalil Raouf A.
Published: February 2004

Certain forms of coronary artery disease do not respond to treatment with Ca
2+
channel blockers, and a role for endothelin-1 (ET-1) in Ca
2+
antagonist-insensitive forms of coronary vasospasm has been suggested; however, the signaling mechanisms involved are unclear. We tested the hypothesis that a component of ET-1–induced coronary smooth muscle contraction is Ca
2+
antagonist-insensitive and involves activation of protein kinase C (PKC). Cell contraction was measured in smooth muscle cells isolated from porcine coronary artery, [Ca
2+
]
i
was measured in fura-2 loaded cells, and the cytosolic and particulate fractions were examined for PKC activity and reactivity with isoform-specific PKC antibodies using Western blot analysis. In Hank’s solution (1 mmol/L Ca
2+
), ET-1 (10
−7
mol/L) caused a transient increase in [Ca
2+
]
i
(236±14 nmol/L) followed by a maintained increase in [Ca
2+
]
i
(184±8 nmol/L) and 35% cell contraction. The Ca
2+
channel blockers verapamil and diltiazem (10
−6
mol/L) abolished the maintained ET-1–induced [Ca
2+
]
i
, but only partially inhibited ET-1–induced cell contraction to 18%. The verapamil-insensitive component of ET-1 contraction was inhibited by the PKC inhibitors calphostin C and ε-PKC
V1–2
. ET-1 caused translocation of Ca
2+
-dependent α-PKC and Ca
2+
-independent ε-PKC from the cytosolic to the particulate fraction that was inhibited by calphostin C. Verapamil abolished ET-1–induced translocation of α-PKC, but not that of ε-PKC. Phorbol 12-myristate 13-acetate (10
−6
mol/L), a direct activator of PKC, caused 22% cell contraction, with no increase in [Ca
2+
]
i
, and translocation of ε-PKC that was inhibited by calphostin C, but not by verapamil. KCl (51 mmol/L), which stimulates Ca
2+
influx, caused 35% cell contraction and increase in [Ca
2+
]
i
(291±11 nmol/L) that were inhibited by verapamil, but not by calphostin C, and did not cause translocation of α- or ε-PKC. In Ca
2+
-free (2 mmol/L EGTA) Hank’s solution, ET-1 caused 15% cell contraction, with no increase in [Ca
2+
]
i
, and translocation of ε-PKC that were inhibited by ε-PKC V1–2 inhibitory peptide. Thus, a significant component of ET-1–induced contraction of coronary smooth muscle is Ca
2+
antagonist-insensitive and involves activation and translocation of Ca
2+
-independent ε-PKC, and may represent a signaling mechanism of Ca
2+
antagonist-resistant forms of coronary vasospasm.
Causes and Consequences of Increased Sympathetic Activity in Renal Disease
Authors: Joles Jaap A. , Koomans Hein A.
Published: February 2004

Much evidence indicates increased sympathetic nervous activity (SNA) in renal disease. Renal ischemia is probably a primary event leading to increased SNA. Increased SNA often occurs in association with hypertension. However, the deleterious effect of increased SNA on the diseased kidney is not only caused by hypertension. Another characteristic of renal disease is unbalanced nitric oxide (NO) and angiotensin (Ang) activity. Increased SNA in renal disease may be sustained because a state of NO–Ang II unbalance is also present in the hypothalamus. Very few studies have directly compared the efficacy of adrenergic blockade with other renoprotective measures. Third-generation β-blockers seem to have more protective effects than traditional β-blockers, possibly via stimulation of NO release. Although it has been extensively documented that muscle SNA is increased in chronic renal failure, data on renal SNA and cardiac SNA are not available for these patients before end-stage renal disease. It is also unknown whether additional treatment with third-generation β-blockers can delay the progression of renal injury and prevent cardiac injury in chronic renal failure more efficiently than conventional treatment with angiotensin-converting enzyme inhibitors only.
Salt Supplementation Improves Orthostatic Cerebral and Peripheral Vascular Control in Patients With Syncope
Authors: Claydon Victoria E. , Hainsworth Roger
Published: February 2004

Salt supplementation improves orthostatic tolerance in many patients with posturally related syncope (PRS). This study aimed to examine whether in those patients who responded to salt loading there was also evidence of improved cerebral autoregulation and more powerful peripheral vasoconstriction during orthostasis. Eleven PRS patients were studied before and after ingestion of 100 mmol/d slow sodium for 2 months. Subjects underwent an orthostatic stress test of combined head-up tilting and lower body suction. We continuously monitored heart rate (ECG), blood pressure (Finapres), forearm and cerebral blood flow velocities (Doppler ultrasound), and end-tidal carbon dioxide (CO
2
). Forearm vascular resistance was calculated from pressure divided by velocity. Cerebral autoregulation was assessed from the correlation coefficient of the relationship between cerebral blood pressure and velocity. Salt loading had no effect on resting heart rate or blood pressure. Symptoms and orthostatic tolerance significantly improved in 10 of the patients. This was associated with a significant increase in the maximal forearm vasoconstriction from 64.4%±13.7% (SEM) to 135.2%±23.9% (
P
<0.005). The relationship between cerebral velocity and pressure was less strong (before salt:
r
=0.74±0.8; after salt:
r
=0.41±0.1;
P
<0.02), indicating improved autoregulation. End-tidal CO
2
levels were not different between the 2 tests. Salt loading in PRS patients increases orthostatic tolerance and improves cerebrovascular and peripheral vascular control without affecting blood pressures. These changes are likely to contribute to the beneficial effects of salt loading in these patients.
Mild to Moderate Hypothermia Prevents Microvascular Basal Lamina Antigen Loss in Experimental Focal Cerebral Ischemia
Authors: Hamann Gerhard F. , Burggraf Dorothe , Martens Helge K. , Liebetrau Martin , Jäger Gabriele , Wunderlich Nathalie , DeGeorgia Michael , Krieger Derk W.
Published: February 2004

Background and Purpose—

Microvascular basal lamina damage occurs after cerebral ischemia and is important for the development of hemorrhage. The aim of this study was to determine whether hypothermia could maintain microvascular integrity in ischemic stroke.

Methods—

Using the suture model, we subjected 12 rats to 3 hours of focal ischemia and 24 hours of reperfusion. Six rats received postischemic normothermia (37°C) and 6 received hypothermia (32°C to 34°C) for the reperfusion period; a group of 6 sham-operated animals without ischemia was used as control. Collagen type IV and hemoglobin were measured by Western blot analysis, matrix metalloproteinase (MMP)-2 and MMP-9 by gelatin zymography, and urokinase-type plasminogen activator (uPA) and tissue-type plasminogen activator (tPA) by plasminogen-casein zymography.

Results—

Hypothermia reduced basal lamina collagen type IV loss: 87±16% (hypothermia) versus 43±4% (normothermia) in basal ganglia and 74±16% versus 64±4% in cortex; hypothermia reduced hemorrhage from 431±65% (normothermia) to 241±28% (basal ganglia) (
P
<0.05). Hypothermia also reduced MMP-2, MMP-9, uPA, and tPA (basal ganglia: MMP-2: 71±20% [hypothermia] versus 109±3% [normothermia]; MMP-9: 38±12% versus 115±4%; uPA activity: 310±86% versus 1019±22%; tPA activity: 61±17% versus 111±13%; cortex: MMP-2: 53±6% versus 116±1%; MMP-9: 16±4% versus 123±3%; uPA: 180±27% versus 176±10%; tPA: 91±15% versus 101±8%; each difference:
P
<0.001) (nonischemic control side=100%).

Conclusions—

Hypothermia maintains microvascular integrity and reduces hemorrhage and the activities of MMP-2, MMP-9, uPA, and tPA.
Potassium Channel Dysfunction in Cerebral Arteries of Insulin-Resistant Rats Is Mediated by Reactive Oxygen Species
Authors: Erdös Benedek , Simandle Steve A. , Snipes James A. , Miller Allison W. , Busija David W.
Published: February 2004

Background and Purpose—

Insulin resistance (IR) increases the risk of stroke in humans. One possible underlying factor is cerebrovascular dysfunction resulting from altered K
+
channel function. Thus, the goal of this study was to examine K
+
channel–mediated relaxation in IR cerebral arteries.

Methods—

Experiments were performed on pressurized isolated middle cerebral arteries (MCAs) from fructose-fed IR and control rats.

Results—

Dilator responses to iloprost, which are BK
Ca
channel mediated, were reduced in the IR compared with control arteries (19±2% versus 33±2% at 10
−6
mol/L). Similarly, relaxation to the K
ATP
opener pinacidil was diminished in the IR MCAs (17±2%) compared with controls (38±2% at 10
−5
mol/L). IR also reduced the K
ATP
channel–dependent component in calcitonin gene-related peptide–induced dilation; however, the magnitude of the relaxation remained unchanged in IR because of a nonspecified K
+
channel–mediated compensatory mechanism. In contrast, K
ir
channel–mediated relaxation elicited by increases in extracellular [K
+
] (4 to 12 mmol/L) was similar in the control and IR arteries. Blockade of the K
ir
and K
v
channels with Ba
2+
and 4-aminopyridine, respectively, constricted the MCAs in both experimental groups with no significant difference. Pretreatment of arteries with superoxide dismutase (200 U/mL) plus catalase (150 U/mL) restored the dilatory responses to iloprost and pinacidil in the IR arteries. Immunoblots showed that the expressions of the pore-forming subunits of the examined K
+
channels are not altered by IR.

Conclusions—

IR induces a type-specific K
+
channel dysfunction mediated by reactive oxygen species. The alteration of K
ATP
and BK
Ca
channel–dependent vascular responses may be responsible for the increased risk of cerebrovascular events in IR.
Diffusion-Weighted Magnetic Resonance Imaging and Neurobiochemical Markers After Aortic Valve Replacement
Authors: Stolz Erwin , Gerriets Tibo , Kluge Alexander , Klövekorn Wolf-Peter , Kaps Manfred , Bachmann Georg
Published: February 2004

Background and Purpose—

Cardiac surgery carries a high risk of neurological complications; therefore, these patients would be an appropriate target population for neuroprotective strategies. In this study, we evaluated postoperative diffusion-weighted imaging (DWI) as a potential surrogate marker for brain embolism and its relationship to neurobiochemical markers of brain injury.

Methods—

Of a total of 45 consecutive patients undergoing aortic valve replacement, 37 completed preoperative and postoperative MRI. At the time of the MRI studies, serum S100β and neuron-specific enolase concentrations were determined. Preexisting T2 and postoperative DWI lesion volumes were quantified. All patients had a blinded neurological examination before and after operation.

Results—

New perioperative DWI lesions were present in 14 patients (38%), of whom only 3 developed focal neurological deficits. Eighteen small lesions were found in the white matter or vascular border zones in all but 2 patients with territorial stroke. The appearance of new DWI lesions correlated with age, pre-existing T2 lesion volume, and postoperative S100β concentrations on days 2 to 4 after surgery. In a forward stepwise canonical discrimination model, only T2 lesion volume was selected as a relevant variable.

Conclusions—

The incidence of postoperative DWI lesions in aortic valve replacement is high, and a suitable marker for neuroprotective trials would be a reduction in the number of such lesions. The volume of preexisting T2 lesions is related to the development of perioperative DWI lesions.
Cerebral Hemodynamics During the Valsalva Maneuver
Authors: Zhang Rong , Crandall Craig G. , Levine Benjamin D.
Published: February 2004

Background and Purpose—

The aim of this study was to differentiate the mechanical effects of the Valsalva maneuver (VM) from the effects of changes in autonomic neural activity on cerebral hemodynamics in humans.

Methods—

Nine healthy subjects performed the VM before and after autonomic ganglionic blockade with trimethaphan. Blood pressure (BP) was measured in the radial artery with an indwelling catheter or at the finger by Finapres. Cerebral blood flow (CBF) velocity was measured in the middle cerebral artery with transcranial Doppler; end-tidal CO
2
was measured by mass spectrometry.

Results—

Before blockade, during phase II of the VM, BP was reduced by 27% and CBF velocity was reduced by 33% (magnitude of changes during phase II divided by baseline measurements before the VM,
P
<0.05). Cerebrovascular conductance index (CVCI) increased by 21%. During phase IV, overshoot of CBF velocity was proportionately greater than that of BP (46% versus 30%). After blockade, during phase II, BP fell to a much greater degree by 50%, while CBF velocity decreased even more by 60% associated with an increase in CVCI by 33%. During phase IV, despite the absence of BP overshoot, CBF velocity still increased by 55% and CVCI by 33%. Both were significantly greater than before blockade.

Conclusions—

After ganglionic blockade, cerebral autoregulation is unable to prevent the substantial fall in CBF induced by the marked reduction in BP during the VM. Enhanced phase IV increases in both CBF velocity and CVCI reflect the intrinsic characteristics of cerebral hyperemic responses, which are likely modified in part by the removal of vasoconstrictor effects of autonomic neural activity elicited during the VM.
The interferon regulatory factor ICSBP/IRF-8 in combination with PU.1 up-regulates expression of tumor suppressor p15Ink4b in murine myeloid cells
Authors: Schmidt Martina , Bies Juraj , Tamura Tomohiko , Ozato Keiko , Wolff Linda
Published: February 2004

CDKN2B (INK4B), which encodes the cyclin-dependent kinase inhibitor p15INK4b, is up-regulated by many cytokines found in hematopoietic environments in vivo. In human acute myeloid leukemias (AMLs), it is inactivated with high frequency. To gain insight into the regulatory pathways leading to the normal activation of p15Ink4b expression, we examined interferon β (IFNβ)–induced transcription. Using reporter gene assays in murine myeloid cells M1, we determined that a 328-bp fragment, located 117 to 443 bp upstream of the translation initiation site, was sufficient to activate transcription. Both the interferon consensus sequence-binding protein/interferon regulatory factor 8 (ICSBP/IRF-8) and PU.1 were able to increase transcription from this region. It was determined that both ICSBP and PU.1 must bind to DNA to form a stable PU.1/ICSBP binding complex. Interestingly, introduction of the ICSBP into ICSBP-null Tot2 cells led to a significant increase in p15Ink4b RNA expression. This regulation of the Ink4b promoter is apparently myeloid specific because both ICSBP and PU.1 are myeloid commitment factors. Importantly, this provides a mechanism to explain in part the tumor suppressor activity of ICSBP, since ICSBP-deficient mice develop a chronic myelogenous leukemia (CML)–like disease and a high percentage of human AML and CML lack ICSBP transcripts.
Mechanisms associated with IL-6–induced up-regulation of Jak3 and its role in monocytic differentiation
Authors: Mangan James K. , Rane Sushil G. , Kang Anthony D. , Amanullah Arshad , Wong Brian C. , Reddy E. Premkumar
Published: February 2004

We report here that Janus kinase 3 (Jak3) is a primary response gene for interleukin-6 (IL-6) in macrophage differentiation, and ectopic overexpression of Jak3 accelerates monocytic differentiation of normal mouse bone marrow cells stimulated with cytokines. Furthermore, we show that incubation of normal mouse bone marrow cells with a JAK3-specific inhibitor results in profound inhibition of myeloid colony formation in response to granulocyte-macrophage colony-stimulating factor or the combination of stem cell factor, IL-3, and IL-6. In addition, mutagenesis of the Jak3 promoter has revealed that Sp1 binding sites within a -67 to -85 element and a signal transducer and activator of transcription (Stat) binding site at position -44 to -53 are critical for activation of Jak3 transcription in murine M1 myeloid leukemia cells stimulated with IL-6. Electrophoretic mobility shift assay (EMSA) analysis has demonstrated that Sp1 can bind to the -67 to -85 element and Stat3 can bind to the -44 to -53 STAT site in IL-6-stimulated M1 cells. Additionally, ectopic overexpression of Stat3 enhanced Jak3 promoter activity in M1 cells. This mechanism of activation of the murine Jak3 promoter in myeloid cells is distinct from a recently reported mechanism of activation of the human JAK3 promoter in activated T cells.
Hematopoietic stem cell dose correlates with the speed of immune reconstitution after stem cell transplantation
Authors: Chen Benny J. , Cui Xiuyu , Sempowski Gregory D. , Domen Jos , Chao Nelson J.
Published: February 2004

In the current study, we tested whether higher numbers of hematopoietic stem cells correlate with the speed of immune reconstitution in a congenic transplantation model (C57BL/Ka, CD45.1, Thy1.1→C57BL/6, CD45.2, Thy1.2) using purified hematopoietic stem cells (c-Kit+Thy1.1lowLin-/lowSca-1+). There were 3 different doses of stem cells used (400, 1000, and 5000). Phenotypic analyses in peripheral blood and spleen demonstrated that higher numbers of infused stem cells are associated with more rapid regeneration of T cells (CD4+, CD8+, naive CD4+, naive CD8+) and B cells at early time points. The numbers of T and B cells eventually became equivalent between different dose groups at late time points. Production of interleukin-2 and inter-feron-γ per T cell was similar regardless of stem cell dose even when tested at the time when there were significant differences in peripheral T-cell counts. The improved immune recovery was attributed to a more rapid regeneration of donor-type immune cells. Higher numbers of total thymocytes and signal joint T-cell receptor excision circles were observed in the higher dose stem cell recipients, suggesting that accelerated regeneration of T cells was due to enhanced thymopoiesis. (Blood. 2004;103:4344-4352)
Caspase-3 has a nonapoptotic function in erythroid maturation
Authors: Carlile Graeme W. , Smith Deborah H. , Wiedmann Martin
Published: February 2004

Caspase-3 plays a central role in apoptosis. It is also activated in normal erythropoiesis, with its activity peaking early during development (erythroid colony-forming unit [CFU-E] stage). In the present study, we have reduced the expression and subsequent enzymatic activity of caspase-3 by transfection of small interfering RNA (siRNA) directed to caspase-3 in a differentiating human erythroid culture system. We find that siRNA treatment yields a 50% reduction in cells that undergo enucleation with no change in the fraction of cells that undergo apoptosis, measured throughout the culture. Furthermore, a substantial fraction of treated cells are unable to complete the transition from pronormoblasts to basophilic normoblasts. These results demonstrate that caspase-3 is required for efficient erythropoiesis in this model system. (Blood. 2004;103:4310-4316)
Deletion of the alternatively spliced fibronectin EIIIA domain in mice reduces atherosclerosis
Authors: Tan Michelle H. , Sun Zhengwu , Opitz Sarah L. , Schmidt Tracy E. , Peters John H. , George Elizabeth L.
Published: February 2004

The alternatively spliced and highly conserved EIIIA domain of fibronectin (FN) is included in most FN of the extracellular matrix in embryos. In adults, both extracellular matrix and plasma FN essentially lack EIIIA. In diverse inflammatory situations however, EIIIA is specifically included by regulated RNA splicing. In atherosclerotic lesions, FN, including the EIIIA domain (EIIIA-FN), is abundant, whereas FN in the flanking vessel wall lacks EIIIA. Lesional EIIIA-FN is localized with endothelial cells and macrophage foam cells. To directly test the function of EIIIA-FN, we generated EIIIA-null (EIIIA–/–) mice that lack the EIIIA exon and crossed them with apolipoprotein E (ApoE)–null (ApoE–/–) mice that develop arterial wall lesions. Compared with ApoE–/– controls, EIIIA–/–ApoE–/– mice had significantly smaller lesions throughout the aortic tree. EIIIA-FN was increased in ApoE–/– plasma, and total plasma cholesterol was reduced in EIIIA–/–ApoE–/– mice, specifically in large lipoprotein particles, suggesting a functional role for plasma EIIIA-FN. To assess a role for macrophage EIIIA-FN in the vessel wall, we conducted in vitro foam cell assays. EIIIA–/–ApoE–/– macrophages accumulated significantly less intracellular lipid than control ApoE–/– cells. These results provide genetic evidence that suggests roles for EIIIA-FN in plasma lipoprotein metabolism and in foam cell formation.
Myeloid-biased hematopoietic stem cells have extensive self-renewal capacity but generate diminished lymphoid progeny with impaired IL-7 responsiveness
Authors: Muller-Sieburg Christa E. , Cho Rebecca H. , Karlsson Lars , Huang Jing-F. , Sieburg Hans B.
Published: February 2004

The adult hematopoietic stem cell (HSC) compartment contains a substantial population of lineage-biased (Lin-bi) HSCs. Lin-bi HSCs generate cells of all hematopoietic lineages, albeit with skewed ratios of lymphoid to myeloid cells. The biased ratios are stable through serial transplantation, demonstrating that lineage bias is an inherent function of the HSCs. To define the mechanisms that cause lineage bias, the developmental potential of myeloid-biased (My-bi) HSCs was characterized. In serial transplantation experiments, My-bi HSCs contributed significantly longer to repopulation than other types of HSCs. The long lifespan indicates that My-bi HSCs are important for the persistence of HSC function throughout life. My-bi HSCs produce normal levels of myeloid precursors but reduced levels of precursors for the T- and B- lymphocyte lineages. Gene array analysis suggested that the lymphoid progeny of My-bi HSCs express lowered levels of interleukin-7 (IL-7) receptor. Indeed, the progeny derived from My-bi HSCs failed to respond to IL-7 in vitro. Thus, My-bi HSCs are programmed for diminished lymphopoiesis through a mechanism that involves a blunted response of its progeny to the central lymphokine IL-7. The data demonstrate that epigenetic regulation on the level of the HSCs can directly affect the number, composition, and function of the mature progeny.
Impaired APC cofactor activity of factor V plays a major role in the APC resistance associated with the factor V Leiden (R506Q) and R2 (H1299R) mutations
Authors: Castoldi Elisabetta , Brugge Jeroen M. , Nicolaes Gerry A. F. , Girelli Domenico , Tans Guido , Rosing Jan
Published: February 2004

Activated protein C (APC) resistance is a major risk factor for venous thrombosis. Factor V (FV) gene mutations like FVLeiden (R506Q) and FVR2 (H1299R) may cause APC resistance either by reducing the susceptibility of FVa to APC-mediated inactivation or by interfering with the cofactor activity of FV in APC-catalyzed FVIIIa inactivation. We quantified the APC cofactor activity expressed by FVLeiden and FVR2 and determined the relative contributions of reduced susceptibility and impaired APC cofactor activity to the APC resistance associated with these mutations. Plasmas containing varying concentrations of normal FV, FVLeiden, or FVR2 were assayed with an APC resistance assay that specifically measures the APC cofactor activity of FV in FVIIIa inactivation, and with the activated partial thromboplastin time (aPTT)-based assay, which probes both the susceptibility and APC cofactor components. FVR2 expressed 73% of the APC cofactor activity of normal FV, whereas FVLeiden exhibited no cofactor activity in FVIIIa inactivation. Poor susceptibility to APC and impaired APC cofactor activity contributed equally to FVLeiden-associated APC resistance, whereas FVR2-associated APC resistance was entirely due to the reduced APC cofactor activity of FVR2. Thrombin generation assays confirmed the importance of the anticoagulant activity of FV and indicated that FVLeiden homozygotes are exposed to a higher thrombotic risk than heterozygotes because their plasma lacks normal FV acting as an anticoagulant protein.
A single recombinant anti-RhD IgG prevents RhD immunization: association of RhD-positive red blood cell clearance rate with polymorphisms in the FcγRIIA and FcγIIIA genes
Authors: Miescher Sylvia , Spycher Martin O. , Amstutz Hanspeter , de Haas Masja , Kleijer Marion , Kalus Ulrich J. , Radtke Hartmut , Hubsch Alphonse , Andresen Irmgard , Martin Roland M. , Bichler Johann
Published: February 2004

A single recombinant immunoglobulin G1 (IgG1) anti-RhD antibody (MonoRho) was compared with a currently used polyclonal anti-RhD product (Rhophylac) in a phase 1 study for safety, efficacy of Rhesus D (RhD)–positive red blood cell (RBC) clearance, and prevention of RhD immunization in RhD-negative men challenged with 15 mL RhD-positive RBCs. Both the polyclonal product and recombinant anti-RhD effectively cleared RhD-positive RBCs after intravenous and intramuscular injection. The recombinant anti-RhD demonstrated a slower clearance rate compared with the polyclonal anti-RhD. There was no dose response, and there was considerable variation among subjects who received the same dose of recombinant anti-RhD. Interestingly, RhD-positive RBC clearance rates were strongly associated with Fcγ receptor IIA (FcγRIIA) and FcγIIIA but not with FcγIIIB polymorphisms. Subjects homozygous for FcγRIIA-131H or FcγRIIIA-158V allotypes showed a faster clearance rate compared with both the heterozygote and the corresponding alternative homozygote allotypes. A similar but less marked trend was seen for the polyclonal anti-RhD. Despite the variation in clearance rates there was no evidence of anti-RhD alloantibodies in any of the subjects at +6 months after the RBC challenge.
Epitope mapping of ADAMTS13 autoantibodies in acquired thrombotic thrombocytopenic purpura
Authors: Klaus Christoph , Plaimauer Barbara , Studt Jan-Dirk , Dorner Friedrich , Lämmle Bernhard , Mannucci Pier Mannuccio , Scheiflinger Friedrich
Published: February 2004

Severe deficiency of the von Willebrand factor (VWF)-cleaving protease ADAMTS13 can lead to thrombotic thrombocytopenic purpura (TTP), a disease associated with the widespread formation of platelet-rich thrombi in many organs. Autoantibodies that inactivate ADAMTS13 are the most frequent cause of acquired TTP. Little is known about epitope specificity and reactivity of anti-ADAMTS13 antibodies. In this study, a series of ADAMTS13 domains were expressed in Escherichia coli, and the reactivity of purified recombinant fragments with anti-ADAMTS13 auto-antibodies from 25 patients with severe ADAMTS13 deficiency was evaluated in vitro. All TTP plasmas contained antibodies directed against the cysteine-rich spacer (cys-rich/spacer) domain of ADAMTS13. In the plasma of 3 patients, antibodies were detected that reacted exclusively with the cys-rich/spacer domain, underscoring the importance of this region for functional activity of ADAMTS13. In 64% of the plasmas, antibodies reacted with the 2 CUB domains, and in 56% they reacted with the isolated first thrombospondin type 1 (TSP-1) repeat and with the compound fragment consisting of the catalytic, the disintegrin-like, and the TSP1-1 domain. Less frequently, in 28% of the plasmas, antibodies reacted with the TSP1 repeats 2 to 8. Unexpectedly, antibodies reacted with the propeptide region in 20% of the plasmas. In conclusion, this study shows that even though anti-ADAMTS13 autoantibodies react with multiple domains of the protease, the cys-rich/spacer domain is consistently involved in antibody reactivity. (Blood. 2004;103:4514-4519)
Discrimination and classification of Eurycoma longifolia Jack in medicinal foods by means of a DSP-based electronic taste sensor
Authors: Abdullah M. Z. , Rahman A. S.A. , Shakaff A. Y.M. , Noor A. M.
Published: February 2004

Eurycoma longifolia Jack (ELJ), better known as Malaysian Ginseng, is fast becoming popular due to its efficacy as a general health and testosterone booster, and for its known aphrodisiac properties. With the growing popularity of ELJ, together with the increasing consumer demand for healthy foods, it is expected that the number of healthy food suppliers boasting claims that their products are enriched with this medicinal herb will also increase. It is, therefore, important to device a strategy that can rapidly provide corroborating evidence in order to assess the authenticity of such a claim onsite. A portable electronic taste sensor employing digital signal processing (DSP) technology was developed to meet this objective. The sensor was constructed from eight kinds of lipid membranes forming the multichannel artificial taste-sensing equipment. The system was trained to detect ELJ into four different concentrations: 0.01%, 0.03%, 0.05% and 0.08%. Over 100 samples were tested, from which the taste system was able to correctly classify the test mixtures at a greater than 90% success rate. This classification success rate, however, dropped to well below 50% when data from multichannel were used independently, indicating the between-channel variations were relatively small. Data fusion via principal component analysis helped enlarge the variation with the first and second principal components, contributed nearly 92% of the total variance. Equipped with 32-bit state-of-the-art DSPs, the taste system allowed both training and testing schemes to be implemented directly onboard.
Combined Effects of p53, p21, and pRb Expression in the Progression of Bladder Transitional Cell Carcinoma
Authors: Chatterjee Sunanda J. , Datar Ram , Youssefzadeh David , George Ben , Goebell Peter J. , Stein John P. , Young Lillian , Shi Shan-Rong , Gee Conway , Groshen Susan , Skinner Donald G. , Cote Richard J.
Published: February 2004

Purpose

To determine the combined effects of p53, p21, and pRb alterations in predicting the progression of bladder transitional cell carcinoma.

Patients and Methods

p53, p21, and pRb expression was examined immunohistochemically on archival radical cystectomy samples from 164 patients with invasive or high-grade recurrent superficial transitional cell carcinoma (TCC; lymph node–negative, 117 patients; lymph node–positive, 47 patients). Median follow-up was 8.6 years. Based on percentage of nuclear reactivity, p53 was considered as wild-type (0% to 10%) or altered (> 10%); p21 was scored as wild-type (>10%) or altered (< 10%); and pRb status was considered wild-type (1% to 50%) or altered (0% or > 50%).

Results

As individual determinants, the p53, p21, and pRb status were independent predictors of time to recurrence (P < .001, P < .001, and P < .001, respectively), and overall survival (P < .001, P = .002, and P = .001, respectively). By examining these determinants in combination, patients were categorized as group I (no alteration in any determinant, 47 patients), group II (any one determinant altered, 51 patients), group III (any two determinants altered, 42 patients), and group IV (all three determinants altered, 24 patients). The 5-year recurrence rates in these groups were 23%, 32%, 57%, and 93%, respectively (log-rank P < .001), and the 5-year survival rates were 70%, 58%, 33%, and 8%, respectively (log-rank P < .001). After stratifying by stage, the number of altered proteins remained significantly associated with time to recurrence and overall survival.

Conclusion

This study suggests that alterations in p53, p21, and pRb act in cooperative or synergistic ways to promote bladder cancer progression. Examining these determinants in combination provides additional information above the use of a single determinant alone.
Hormone Replacement Therapy and Life Expectancy After Prophylactic Oophorectomy in Women With BRCA1/2 Mutations: A Decision Analysis
Authors: Armstrong Katrina , Schwartz J. Sanford , Randall Thomas , Rubin Stephen C. , Weber Barbara
Published: February 2004

Purpose

The decision about prophylactic oophorectomy is difficult for many premenopausal women with BRCA1/2 mutations because of concerns and controversy about the use of hormone replacement therapy (HRT) after oophorectomy.

Patients and Methods

A Markov decision analytic model used the most current epidemiologic data to assess the expected outcomes of prophylactic oophorectomy with or without HRT (to age 50 years or for life) in cohorts of women with BRCA1/2 mutations. Sensitivity analyses were conducted to assess the impact of alternative assumptions about effects of HRT, effects of prophylactic oophorectomy, and risks of cancer associated with BRCA1/2 mutations.

Results

In our model, prophylactic oophorectomy lengthened life expectancy in women with BRCA1/2 mutations, irrespective of whether HRT was used after oophorectomy. This gain ranged from 3.34 to 4.65 years, depending on age at oophorectomy. Use of HRT after oophorectomy was associated with relatively small changes in life expectancy (+0.17 to −0.34 years) when HRT was stopped at age 50, but larger decrements in life expectancy if HRT was continued for life (−0.79 to −1.09 years). HRT was associated with a gain in life expectancy of between 0.39 and 0.79 years for mutation carriers undergoing both prophylactic mastectomy and oophorectomy.

Conclusion

On the basis of the results of this decision analysis, we recommend that women with BRCA1/2 mutations undergo prophylactic oophorectomy after completion of childbearing, decide about short-term HRT after oophorectomy based largely on quality-of-life issues rather than life expectancy, and, if using HRT, consider discontinuing treatment at the time of expected natural menopause, approximately age 50 years.
Interleukin-2- and Interferon Alfa-2a-Based Immunochemotherapy in Advanced Renal Cell Carcinoma: A Prospectively Randomized Trial of the German Cooperative Renal Carcinoma Chemoimmunotherapy Group (DGCIN)
Authors: Atzpodien J. , Kirchner H. , Jonas U. , Bergmann L. , Schott H. , Heynemann H. , Fornara P. , Loening S.A. , Roigas J. , Müller S.C. , Bodenstein H. , Pomer S. , Metzner B. , Rebmann U. , Oberneder R. , Siebels M. , Wandert T. , Puchberger T. , Reitz M.
Published: February 2004

Purpose

We conducted a prospectively randomized clinical trial to compare the efficacy of three outpatient therapy regimens in 341 patients with progressive metastatic renal cell carcinoma.

Patients and Methods

Patients were stratified according to known clinical predictors and were subsequently randomly assigned. Treatment arms were: arm A (n = 132), subcutaneous interferon alfa-2a (sc-IFN-α-2a), subcutaneous interleukin-2 (sc-IL-2), and intravenous (IV) fluorouracil; arm B (n = 146): arm A treatment combined with per oral 13-cis-retinoic acid; and arm C (n = 63), sc-IFN-α-2a and IV vinblastine.

Results

Treatment (according to the standard 8-week Hannover Atzpodien regimen) arms A, B, and C yielded objective response rates of 31%, 26%, and 20%, respectively. Arm B, but not arm A, showed a significantly improved progression-free survival (PFS) compared with arm C (P = .0248). Both arm A (median overall survival, 25 months; P = .0440) and arm B (median overall survival, 27 months; P = .0227) led to significantly improved overall survival (OS) compared with arm C (median OS, 16 months). All three sc-IFN-α-2a–based therapies were moderately or well tolerated.

Conclusion

Our results established the safety and improved long-term therapeutic efficacy of sc-IL-2 plus sc-INF-α-2a–based outpatient immunochemotherapies, compared with sc-INF-α-2a/IV vinblastine.
Uterine Msx-1 and Wnt4 Signaling Becomes Aberrant in Mice with the Loss of Leukemia Inhibitory Factor or Hoxa-10: Evidence for a Novel Cytokine-Homeobox-Wnt Signaling in Implantation
Authors: Daikoku Takiko , Song Haengseok , Guo Yong , Riesewijk Anne , Mosselman Sietse , Das Sanjoy K. , Dey Sudhansu K.
Published: February 2004

Successful implantation absolutely depends on the reciprocal interaction between the implantation-competent blastocyst and the receptive uterus. Expression and gene targeting studies have shown that leukemia inhibitory factor (LIF), a cytokine of the IL-6 family, and Hoxa-10, an abdominalB-like homeobox gene, are crucial to implantation and decidualization in mice. Using these mutant mice, we sought to determine the importance of Msx-1 (another homeobox gene formerly known as Hox-7.1) and of Wnt4 (a ligand of the Wnt family) signaling in implantation because of their reported functions during development. We observed that Msx-1, Wnt4, and a Wnt antagonist sFRP4 are differentially expressed in the mouse uterus during the periimplantation period, suggesting their role in implantation. In addition, we observed an aberrant uterine expression of Msx-1 and sFRP4 in Lif mutant mice, and of Wnt4 and sFRP4 in Hoxa-10 mutant mice, further reinforcing the importance of these signaling pathways in implantation. Collectively, the present results provide evidence for a novel cytokine-homeotic-Wnt signaling network in implantation.
Real-Time Detection of Interactions between the Human Oxytocin Receptor and G Protein-Coupled Receptor Kinase-2
Authors: Hasbi Ahmed , Devost Dominic , Laporte Stéphane A. , Zingg Hans H.
Published: February 2004

Although the oxytocin receptor (OTR) mediates many important functions including uterine contractions, milk ejection, and maternal behavior, the mechanisms controlling agonist-induced OTR desensitization have remained unclear, and attempts to demonstrate involvement of a G protein-coupled receptor kinase (GRK) have so far failed. Using the OTR as a model, we demonstrate here directly for the first time the dynamics of agonist-induced interactions of a GRK with a G protein-coupled receptor in real time, using time-resolved bioluminescence resonance energy transfer. GRK2/receptor interactions started within 4 sec, peaked at 10 sec, and decreased to less than 40% within 8 min. By contrast, β-arrestin/OTR interactions initiated only at 10 sec, reached plateau levels at 120 sec, but remained stable with little decrease thereafter. Physical GRK2/OTR association was further demonstrated by coimmunoprecipitation of endogenous GRK2 with activated OTR. In COS-7 cells, which express low levels of GRK2 and β-arrestin, overexpression of GRK2 and β-arrestin increased receptor phosphorylation, desensitization, and internalization to the high levels observed in human embryonic kidney 293 cells. By contrast, specific inhibition of endogenous GRK2 by dominant-negative mutants robustly inhibited OTR phosphorylation and internalization as well as arrestin/OTR interactions. These data characterize the temporal and causal relationship of GRK-2/OTR and β-arrestin/OTR interactions and establish GRK/OTR interaction as a prerequisite for β-arrestin-mediated OTR desensitization.
Reiterated Wnt signaling during zebrafish neural crest development
Authors: Lewis Jessica L. , Bonner Jennifer , Modrell Melinda , Ragland Jared W. , Moon Randall T. , Dorsky Richard I. , Raible David W.
Published: February 2004

While Wnt/β-catenin signaling is known to be involved in the development of neural crest cells in zebrafish, it is unclear which Wnts are involved, and when they are required. To address these issues we employed a zebrafish line that was transgenic for an inducible inhibitor of Wnt/β-catenin signaling, and inhibited endogenous Wnt/β-catenin signaling at discrete times in development. Using this approach, we defined a critical period for Wnt signaling in the initial induction of neural crest,which is distinct from the later period of development when pigment cells are specified from neural crest. Blocking Wnt signaling during this early period interfered with neural crest formation without blocking development of dorsal spinal neurons. Transplantation experiments suggest that neural crest precursors must directly transduce a Wnt signal. With regard to identifying which endogenous Wnt is responsible for this initial critical period, we established that wnt8 is expressed in the appropriate time and place to participate in this process. Supporting a role for Wnt8, blocking its function with antisense morpholino oligonucleotides eliminates initial expression of neural crest markers. Taken together, these results demonstrate that Wnt signals are critical for the initial induction of zebrafish neural crest and suggest that this signaling pathway plays reiterated roles in its development.
Discrete gene sets depend on POU domain transcription factor Brn3b/Brn-3.2/POU4f2 for their expression in the mouse embryonic retina
Authors: Mu Xiuqian , Beremand Phillip D. , Zhao Sheng , Pershad Rashmi , Sun Hongxia , Scarpa Ann , Liang Shuguang , Thomas Terry L. , Klein William H.
Published: February 2004

Brn3b/Brn-3.2/POU4f2 is a POU domain transcription factor that is essential for retinal ganglion cell (RGC) differentiation, axonal outgrowth and survival. Our goal was to establish a link between Brn3b and the downstream events leading to RGC differentiation. We sought to determine both the number and types of genes that depend on Brn3b for their expression. RNA probes from wild-type and Brn3b-/- E14.5, E16.5 and E18.5 mouse retinas were hybridized to a microarray containing 18,816 retina-expressed cDNAs. At E14.5, we identified 87 genes whose expression was significantly altered in the absence of Brn3b and verified the results by real-time PCR and in situ hybridization. These genes fell into discrete sets that encoded transcription factors, proteins associated with neuron integrity and function,and secreted signaling molecules. We found that Brn3b influenced gene expression in non RGCs of the retina by controlling the expression of secreted signaling molecules such as sonic hedgehog and myostatin/Gdf8. At later developmental stages, additional alterations in gene expression were secondary consequences of aberrant RGC differentiation caused by the absence of Brn3b. Our results demonstrate that a small but crucial fraction of the RGC transcriptome is dependent on Brn3b. The Brn3b-dependent gene sets therefore provide a unique molecular signature for the developing retina.
Newborn horizontal cells migrate bi-directionally across the neuroepithelium during retinal development
Authors: Edqvist Per-Henrik D. , Hallböök Finn
Published: February 2004

Cell migration plays an important role during the development of the retina. In this work we have studied the migration of newborn horizontal cells in avian embryonic retina. Using the pattern of the early expressed transcription factors Lim1 and Prox1 we have shown that horizontal cells migrate bi-directionally from their site of birth, close to the ventricular side, to the adjacent (vitreal) side of the neuroepithelium, where they align just next to the prospective ganglion cell layer before migrating back again to their final laminar position in the external part of the inner nuclear layer. The migration occurs between Hamburger and Hamilton stages 24 and 33,which is equivalent to embryonic day 4.5 and 8. Between stages 26 and 30 the horizontal cells reside close to the ganglion cell layer and intra ocular injections of a cytochalasin D, an actin polymerisation blocker that inhibit migration, at stage 29 interfered with the migration of the horizontal cells to their final destination. Furthermore, using biolistic gene transfer with a green fluorescence protein expression vector of retinal slices we were able to record ventricle-directed migration by time-lapse microscopy. Combining biolistics with immunohistochemistry we showed that transfected cells, which have also been translocated in a ventricular direction were positive for the horizontal cell markers Lim1 and Prox1. The alternative path of migration that is described in this work differs from the generally accepted one for horizontal cells and this knowledge will influence the view of how the molecular determination of horizontal cells is specified.
Bmp signals from niche cells directly repress transcription of a differentiation-promoting gene,bag of marbles, in germline stem cells in theDrosophilaovary
Authors: Song Xiaoqing , Wong Marco D. , Kawase Eihachiro , Xi Rongwen , Ding Bee C. , McCarthy John J. , Xie Ting
Published: February 2004

The Drosophila ovary is an attractive system to study how niches control stem cell self-renewal and differentiation. The niche for germline stem cells (GSCs) provides a Dpp/Bmp signal, which is essential for GSC maintenance. bam is both necessary and sufficient for the differentiation of immediate GSC daughters, cystoblasts. Here we show that Bmp signals directly repress bam transcription in GSCs in the Drosophila ovary. Similar to dpp, gbb encodes another Bmp niche signal that is essential for maintaining GSCs. The expression of phosphorylated Mad (pMad), a Bmp signaling indicator, is restricted to GSCs and some cystoblasts, which have repressed bam expression. Both Dpp and Gbb signals contribute to pMad production. bam transcription is upregulated in GSCs mutant for dpp and gbb. In marked GSCs mutant for Med and punt, two essential Bmp signal transducers, bam transcription is also elevated. Finally, we show that Med and Mad directly bind to the bam silencer in vitro. This study demonstrates that Bmp signals maintain the undifferentiated or self-renewal state of GSCs, and directly repress bam expression in GSCs by functioning as short-range signals. Thus, niche signals directly repress differentiation-promoting genes in stem cells in order to maintain stem cell self-renewal.
Preconditioning-induced cytoprotection in hepatocytes requires Ca2+-dependent exocytosis of lysosomes
Authors: Carini Rita , Castino Roberta , De Cesaris Maria Grazia , Splendore Roberta , Démoz Marina , Albano Emanuele , Isidoro Ciro
Published: February 2004

A short period of hypoxia reduces the cytotoxicity produced by a subsequent prolonged hypoxia in isolated hepatocytes. This phenomenon, termed hypoxic preconditioning, is mediated by the activation of adenosine A2A-receptor and is associated with the attenuation of cellular acidosis and Na+ overload normally occurring during hypoxia. Bafilomycin, an inhibitor of the vacuolar H+/ATPase, reverts the latter effects and abrogates the preconditioning-induced cytoprotection. Here we provide evidence that the acquisition of preconditioning-induced cytoprotection requires the fusion with plasma membrane and exocytosis of endosomal-lysosomal organelles. Poisons of the vesicular traffic, such as wortmannin and 3-methyladenine, which inhibit phosphatydilinositol 3-kinase, or cytochalasin D, which disassembles the actin cytoskeleton, prevented lysosome exocytosis and also abolished the preconditioning-associated protection from acidosis and necrosis provoked by hypoxia. Preconditioning was associated with the phosphatydilinositol 3-kinase-dependent increase of cytosolic [Ca2+]. Chelation of free cytosolic Ca2+ in preconditioned cells prevented lysosome exocytosis and the acquisition of cytoprotection. We conclude that lysosome-plasma membrane fusion is the mechanism through which hypoxic preconditioning allows hepatocytes to preserve the intracellular pH and survive hypoxic stress. This process is under the control of phosphatydilinositol 3-kinase and requires the integrity of the cytoskeleton and the rise of intracellular free calcium ions.