bluuebunny/crossref_metadata_2025 huggingface dataset:
Characterization of a pseudoachondroplasia-associated mutation (His587 Arg) in the C-terminal, collagen-binding domain of cartilage oligomeric matrix protein (COMP)
Authors: SPITZNAGEL Luitgard , NITSCHE D. Patric , PAULSSON Mats , MAURER Patrik , ZAUCKE Frank
Published: January 2004

We have introduced a pseudoachondroplasia-associated mutation (His587→Arg) into the C-terminal collagen-binding domain of COMP (cartilage oligomeric matrix protein) and recombinantly expressed the full-length protein as well as truncated fragments in HEK-293 cells. CD spectroscopy revealed only subtle differences in the overall secondary structure of full-length proteins. Interestingly, the mutant COMP did not aggregate in the presence of calcium, as does the wild-type protein. The binding site for collagens was recently mapped to amino acids 579–595 and it was assumed that the His587→Arg mutation influences collagen binding. However full-length mutant COMP bound to collagens I, II and IX, and the binding was not significantly different from that of wild-type COMP. Also a COMP His587→Arg fragment encompassing the calcium-binding repeats and the C-terminal collagen-binding domain bound collagens equally well as the corresponding wild-type protein. The recombinant fragments encompassing the C-terminal domain alone showed multiple bands following SDS/PAGE, although their theoretical molecular masses could be verified by MS. A temperature-induced conformational change was observed in CD spectroscopy, and negative-staining electron microscopy demonstrated that both wild-type and mutant proteins formed defined elongated aggregates after heating to 60 °C. Our results suggest that the His587→Arg mutation is not itself deleterious to the structure and collagen-binding of COMP.
Glucose 6-phosphate causes translocation of phosphorylase in hepatocytes and inactivates the enzyme synergistically with glucose
Authors: AISTON Susan , GREEN Andrew , MUKHTAR Mohammed , AGIUS Loranne
Published: January 2004

The role of glucose 6-P (glucose 6-phosphate) in regulating the activation state of glycogen synthase and its translocation is well documented. In the present study, we investigated the effects of glucose 6-P on the activation state and compartmentation of phosphorylase in hepatocytes. Glucose 6-P levels were modulated in hepatocytes by glucokinase overexpression or inhibition with 5-thioglucose and the effects of AMP were tested using AICAR (5-aminoimidazole-4-carboxamide 1-β-d-ribofuranoside), which is metabolized to an AMP analogue. Inhibition of glucokinase partially counteracted the effect of glucose both on the inactivation of phosphorylase and on the translocation of phosphorylase a from a soluble to a particulate fraction. The increase in glucose 6-P caused by glucokinase overexpression caused translocation of phosphorylase a to the pellet and had additive effects with glucose on inactivation of phosphorylase. It decreased the glucose concentration that caused half-maximal inactivation from 20 to 11 mM, indicating that it acts synergistically with glucose. AICAR activated phosphorylase and counteracted the effect of glucose 6-P on phosphorylase inactivation. However, it did not counteract translocation of phosphorylase by glucose 6-P. Glucose 6-P and AICAR had opposite effects on the activation state of glycogen synthase, but they had additive effects on translocation of the enzyme to the pellet. There was a direct correlation between the translocation of phosphorylase a and of glycogen synthase to the pellet, suggesting that these enzymes translocate in tandem. In conclusion, glucose 6-P causes both translocation of phosphorylase and inactivation, indicating a more complex role in the regulation of glycogen metabolism than can be explained from regulation of glycogen synthase alone.
Molecular basis of the high-affinity activation of type 1 ryanodine receptors by imperatoxin A
Authors: LEE Chul Won , LEE Eun Hui , TAKEUCHI Koh , TAKAHASHI Hideo , SHIMADA Ichio , SATO Kazuki , SHIN Song Yub , KIM D. Han , KIM Jae Il
Published: January 2004

Both imperatoxin A (IpTxa), a 33-residue peptide toxin from scorpion venom, and peptide A, derived from the II–III loop of dihydropyridine receptor (DHPR), interact specifically with the skeletal ryanodine receptor (RyR1), which is a Ca2+-release channel in the sarcoplasmic reticulum, but with considerably different affinities. IpTxa activates RyR1 with nanomolar affinity, whereas peptide A activates RyR1 at micromolar concentrations. To investigate the molecular basis for high-affinity activation of RyR1 by IpTxa, we have determined the NMR solution structure of IpTxa, and identified its functional surface by using alanine-scanning analogues. A detailed comparison of the functional surface profiles for two peptide activators revealed that IpTxa exhibits a large functional surface area (approx. 1900 Å2, where 1 Å=0.1 nm), based on a short double-stranded antiparallel β-sheet structure, while peptide A bears a much smaller functional surface area (approx. 800 Å2), with the five consecutive basic residues (Arg681, Lys682, Arg683, Arg684 and Lys685) being clustered at the C-terminal end of the α-helix. The functional surface of IpTxa is composed of six essential residues (Leu7, Lys22, Arg23, Arg24, Arg31 and Arg33) and several other important residues (His6, Lys8, Arg9, Lys11, Lys19, Lys20, Gly25, Thr26, Asn27 and Lys30), indicating that amino acid residues involved in RyR1 activation make up over the half of the toxin molecule with the exception of cysteine residues. Taken together, these results suggest that the site where peptide A binds to RyR1 belongs to a subset of macrosites capable of being occupied by IpTxa, resulting in differing the affinity and the mode of activation.
Rosiglitazone, an agonist of peroxisome-proliferator-activated receptor gamma (PPARgamma), decreases inhibitory serine phosphorylation of IRS1 in vitro and in vivo
Authors: JIANG Guoqiang , DALLAS-YANG Qing , BISWAS Subarna , LI Zhihua , ZHANG Bei B.
Published: January 2004

Peroxisome-proliferator-activated receptor γ agonists such as rosiglitazone, a thiazolidinedione, improve insulin sensitivity in vivo, but the underlying mechanism(s) remains unclear. Phosphorylation of IRS1 (insulin receptor substrate protein 1) on certain serine residues, including S307 and S612 in rodent IRS1 (equivalent to S312 and S616 in human IRS1), has been shown to play a negative role in insulin signalling. In the present study, we investigated whether rosiglitazone improves insulin sensitivity by decreasing IRS1 inhibitory serine phosphorylation. In HEK-293 (human embryonic kidney 293) cells stably expressing recombinant IRS1 and in 3T3L1 adipocytes, rosiglitazone attenuated PMA-induced IRS1 S307/S612 phosphorylation and decreased insulin-stimulated Akt phosphorylation. We observed increased IRS1 S307 phosphorylation and concomitant decrease in insulin signalling as measured by insulin-stimulated IRS1 tyrosine phosphorylation, and Akt threonine phosphorylation in adipose tissues of Zucker obese rats compared with lean control rats. Treatment with rosiglitazone at 30 mg/kg body weight for 24 and 48 h increased insulin signalling and decreased IRS1 S307 phosphorylation concomitantly. Whereas the 48 h treatment reversed hyper-phosphorylation (and activation) of both c-Jun N-terminal kinase and p38 mitogen-activated protein kinase, the 24 h treatments only decreased hyper-phosphorylation of p38 mitogen-activated protein kinase. The treatment of the Zucker obese rats with rosiglitazone also reversed the high circulating levels of non-esterified fatty acids, which have been shown to be correlated with increased IRS1 serine phosphorylation in other animal models. Taken together, these results suggest that IRS1 inhibitory serine phosphorylation is a key component of insulin resistance and its reversal contributes to the insulin sensitizing effects by rosiglitazone.
Distinct roles for insulin and insulin-like growth factor-1 receptors in pancreatic beta-cell glucose sensing revealed by RNA silencing
Authors: da SILVA XAVIER Gabriela , QIAN Qingwen , CULLEN Peter J. , RUTTER Guy A.
Published: January 2004

The importance of the insulin receptor (IR) and the insulin-like growth factor-1 receptor (IGF-1R) for glucose-regulated insulin secretion and gene expression in pancreatic islet β-cells is at present unresolved. Here, we have used small interfering RNAs (siRNAs) to silence the expression of each receptor selectively in clonal MIN6 β-cells. Reduction of IR levels by >90% completely inhibited glucose (30 mM compared with 3 mM)-induced insulin secretion, but had no effect on depolarization-stimulated secretion. IR depletion also blocked the accumulation of preproinsulin (PPI), pancreatic duodenum homoeobox-1 (PDX-1) and glucokinase (GK) mRNAs at elevated glucose concentrations, as assessed by quantitative real-time PCR analysis (TaqMan®). Similarly, depletion of IGF-1R inhibited glucose-induced insulin secretion but, in contrast with the effects of IR silencing, had little impact on the regulation of gene expression by glucose. Moreover, loss of IGF-1R, but not IR, markedly inhibited glucose-stimulated increases in cytosolic and mitochondrial ATP, suggesting a role for IGF-1R in the maintenance of oxidative metabolism and in the generation of mitochondrial coupling factors. RNA silencing thus represents a useful tool for the efficient and selective inactivation of receptor tyrosine kinases in isolated β-cells. By inhibiting glucose-stimulated insulin secretion through the inactivation of IGF-1R, this approach also demonstrates the existence of insulin-independent mechanisms whereby elevated glucose concentrations regulate PPI, PDX-1 and GK gene expression in β-cells.
The growth hormone receptor interacts with its sheddase, the tumour necrosis factor-alpha-converting enzyme (TACE)
Authors: SCHANTL Julia A. , ROZA Marcel , van KERKHOF Peter , STROUS Ger J.
Published: January 2004

Proteolysis of the GHR (growth hormone receptor) occurs at the cell surface and results in the release of its extracellular domain, the GHBP (growth hormone-binding protein). TACE (tumour necrosis factor-α-converting enzyme) has been identified as a putative protease responsible for GHR cleavage. However, GHR–TACE interaction has not been observed until now. Here, we identified TACE in Chinese hamster cells and confirmed processing and cell-surface expression. Interaction between GHR and TACE was only observed after growth hormone binding. As the growth hormone–GHR2 complex is a poor substrate for TACE, we conclude that the GHR–TACE interaction precedes proteolysis, and is transient. Furthermore, we demonstrate that TACE is present in endosomes, where it partly co-localizes with endocytosed growth hormone ligand.
Differential sorting of tyrosine kinases and phosphotyrosine phosphatases acting on band 3 during vesiculation of human erythrocytes
Authors: MINETTI Giampaolo , CIANA Annarita , BALDUINI Cesare
Published: January 2004

One of the most intensively studied post-translational modifications of erythrocyte proteins is the phosphorylation of tyrosine residues of band 3, which is strictly regulated in vivo by PTKs (protein-tyrosine kinases) and PTPs (protein-phosphotyrosine phosphatases). Two PTKs (p72syk and p56/53lyn) and two PTP activities (PTP1B and SHPTP-2) have been immunologically identified so far in mature human erythrocytes. We have shown previously that band 3 undergoes tyrosine phosphorylation upon a decrease in cell volume, as occurs when erythrocytes treated with Ca2+/Ca2+ ionophore (A23187) lose KCl and release microvesicles. Similar levels of band 3 tyrosine phosphorylation in vesicles and in the parent cells are induced by this treatment. However, we have found that tyrosine phosphorylation of band 3 in vesicles is more stable than in whole erythrocytes. Examination of how the identified PTPs and PTKs are partitioned between the vesicles and the remnant cells during vesiculation reveals that PTP1B, unlike the PTKs, is retained entirely in the parent cell compartment. Since a tight association between PTP1B and band 3 has been documented previously, we have investigated the partitioning of PTP1B and band 3 between the membrane and the membrane-skeletal fractions prepared from resting or Ca2+/A23187-treated cells. Our results rule out the possibility that the preferential retention of PTP1B within the cell was due to an increase in the amount of membrane-skeleton-associated band 3 (and of PTP1B) during the release of spectrin-free vesicles, suggesting a more complex modality of interaction of PTP1B with band 3 in the erythrocyte membrane. Analysis of erythrocytes of different cell ages revealed that PTP1B, unlike the other enzymes examined, was quantitatively conserved during erythrocyte aging. This suggests important roles for the down-regulation of tyrosine phosphorylation of band 3 in erythrocyte physiology, and for vesiculation as a mechanism of human erythrocyte senescence.
Protein tyrosine phosphatase 1B participates in the down-regulation of erythropoietin receptor signalling
Authors: COHEN Jacob , OREN-YOUNG Liat , KLINGMULLER Ursula , NEUMANN Drorit
Published: January 2004

Erythropoietin (EPO) is the principal hormone regulating the proliferation of erythroid precursors and their differentiation into erythrocytes. Binding of ligand to the cell-surface EPO-R (EPO receptor) induces dimerization and JAK2 (Janus kinase 2)-mediated tyrosine phosphorylation of the receptor. Less than 1% of the EPO-Rs are displayed on the cell surface; most of the receptor molecules are retained in intracellular compartments, including the ER (endoplasmic reticulum). Using pervanadate (PV) as a potent tool to inhibit cellular PTPs (protein tyrosine phosphatases), we demonstrated previously the accumulation of mature (endoglycosidase H-resistant) tyrosine-phosphorylated EPO-R [Cohen, Altaratz, Zick, Klingmuller and Neumann (1997) Biochem. J. 327, 391–397]. In the present study, we investigated the participation of the ER-associated PTP1B in the dephosphorylation of intracellular EPO-R. We demonstrate tyrosine phosphorylation of EPO-R in BOSC-23T cells co-expressing EPO-R and the ‘substrate-trapping’ mutant form of PTP1B, PTP1B D181A (referred to as PTP1BD). In vivo interaction between EPO-R and PTP1B suggested that PTP1B dephosphorylates the EPO-R intracellularly. Endoglycosidase H resistance of tyrosine-phosphorylated EPO-R in cells expressing PTP1BD suggested that mature EPO-R is dephosphorylated by PTP1B. Stimulation with EPO of cells co-expressing EPO-R and either PTP1BD or PTP1B resulted in an increase or decrease respectively in phosphotyrosine EPO-R. We thus suggest that PTP1B dephosphorylates EPO-stimulated EPO-R and participates in the down-regulation cascade of EPO-mediated signal transduction.
How versatile are inositol phosphate kinases?
Authors: SHEARS Stephen B.
Published: January 2004

This review assesses the extent and the significance of catalytic versatility shown by several inositol phosphate kinases: the inositol phosphate multikinase, the reversible Ins(1,3,4)P3/Ins(3,4,5,6)P4 kinase, and the kinases that synthesize diphosphoinositol polyphosphates. Particular emphasis is placed upon data that are relevant to the situation in vivo. It will be shown that catalytic promiscuity towards different inositol phosphates is not typically an evolutionary compromise, but instead is sometimes exploited to facilitate tight regulation of physiological processes. This multifunctionality can add to the complexity with which inositol signalling pathways interact. This review also assesses some proposed additional functions for the catalytic domains, including transcriptional regulation, protein kinase activity and control by molecular ‘switching’, all in the context of growing interest in ‘moonlighting’ (gene-sharing) proteins [Jeffery (2003) Ann. Med. 35, 28–35].
Tyr-298 in ephrinB1 is critical for an interaction with the Grb4 adaptor protein
Authors: BONG Yong-Sik , PARK Yeon-Hwa , LEE Hyun-Shik , MOOD Kathleen , ISHIMURA Akihiko , DAAR Ira O.
Published: January 2004

The Eph family of receptor tyrosine kinases and their membrane-bound ligands, the ephrins, are thought to play a role in the regulation of cell adhesion and migration during development by mediating cell-to-cell signalling events. The transmembrane ephrinB protein is a bidirectional signalling molecule that sends a forward signal through the activation of its cognate receptor tyrosine kinase residing on another cell. The reverse signal is transduced into the ephrinB-expressing cell via tyrosine phosphorylation of its conserved C-terminal cytoplasmic domain. Previous work from our laboratory has implicated the activated FGFR1 (fibroblast growth factor receptor 1) as a regulator of a de-adhesion signal that results from overexpression of ephrinB1. In the present study, we report the isolation of Xenopus Grb4 (growth-factor-receptor-bound protein 4), an ephrinB1-interacting protein, and we show that when expressed in Xenopus oocytes, ephrinB1 interacts with Grb4 in the presence of an activated FGFR1. Amino acid substitutions were generated in Grb4, and the resulting mutants were expressed along with ephrinB1 and an activated FGFR in Xenopus oocytes. Co-immunoprecipitation analysis shows that the FLVR motif within the Src homology 2 domain of Xenopus Grb4 is vital for this phosphorylation-dependent interaction with ephrinB1. More importantly, using deletion and substitution analysis we identify the tyrosine residue at position 298 of ephrinB1 as being required for the physical interaction with Grb4, whereas Tyr-305 and Tyr-310 are dispensable. Moreover, we show that the region between amino acids 301 and 304 of ephrinB1 is also required for this critical tyrosine-phosphorylation-dependent event.
In self-defence: hexokinase promotes voltage-dependent anion channel closure and prevents mitochondria-mediated apoptotic cell death
Authors: AZOULAY-ZOHAR Heftsi , ISRAELSON Adrian , ABU-HAMAD Salah , SHOSHAN-BARMATZ Varda
Published: January 2004

In tumour cells, elevated levels of mitochondria-bound isoforms of hexokinase (HK-I and HK-II) result in the evasion of apoptosis, thereby allowing the cells to continue proliferating. The molecular mechanisms by which bound HK promotes cell survival are not yet fully understood. Our studies relying on the purified mitochondrial outer membrane protein VDAC (voltage-dependent anion channel), isolated mitochondria or cells in culture suggested that the anti-apoptotic activity of HK-I occurs via modulation of the mitochondrial phase of apoptosis. In the present paper, a direct interaction of HK-I with bilayer-reconstituted purified VDAC, inducing channel closure, is demonstrated for the first time. Moreover, HK-I prevented the Ca2+-dependent opening of the mitochondrial PTP (permeability transition pore) and release of the pro-apoptotic protein cytochrome c. The effects of HK-I on VDAC activity and PTP opening were prevented by the HK reaction product glucose 6-phosphate, a metabolic intermediate in most biosynthetic pathways. Furthermore, glucose 6-phosphate re-opened both the VDAC and the PTP closed by HK-I. The HK-I-mediated effects on VDAC and PTP were not observed using either yeast HK or HK-I lacking the N-terminal hydrophobic peptide responsible for binding to mitochondria, or in the presence of an antibody specific for the N-terminus of HK-I. Finally, HK-I overexpression in leukaemia-derived U-937 or vascular smooth muscle cells protected against staurosporine-induced apoptosis, with a decrease of up to 70% in cell death. These results offer insight into the mechanisms by which bound HK promotes tumour cell survival, and suggests that its overexpression not only ensures supplies of energy and phosphometabolites, but also reflects an anti-apoptotic defence mechanism.
Time Dependence and Moments of a Family of Time‐Varying Parameter Garch in Mean Models
Authors: Arvanitis Stelios , Demos Antonis
Published: January 2004

Abstract. In this paper we consider the time series dependence, stationarity, and higher moments issues of a family of first‐order conditionally heteroskedastic in mean models with a possibly time‐varying mean parameter. The interest in these models lies in the fact that economic theory and physics often require the connection between the first and second conditional moments of time series. Our results reveal important properties of these models, which are consistent with stylized facts in financial and turbulence data sets. They can also be employed for model identification, estimation, and testing.
Additional cytogenetic abnormalities in adults with Philadelphia chromosome‐positive acute lymphoblastic leukaemia: a study of the Cancer and Leukaemia Group B
Authors: Wetzler Meir , Dodge Richard K. , Mrózek Krzysztof , Stewart Carleton C. , Carroll Andrew J. , Tantravahi Ramana , Vardiman James W. , Larson Richard A. , Bloomfield Clara D.
Published: January 2004

We analysed the nature and prognostic significance of secondary cytogenetic changes in 111 newly diagnosed adults with acute lymphoblastic leukaemia (ALL) and t(9;22)(q34;q11.2) or its variants. Secondary aberrations were seen in 75 (68%) patients. They included, in order of descending frequency: +der(22)t(9;22), +21, abnormalities of 9p, high hyperdiploidy (>50 chromosomes), +8, −7, +X and abnormalities resulting in loss of material from 8p, gain of 8q, gain of 1q and loss of 7p. Eighty patients (72%) had ≥1 normal metaphase in their karyotype. There were four balanced and 12 unbalanced translocations previously unreported in ALL with t(9;22). The t(2;7)(p11;p13) and der(18)t(8;18)(q11.2;p11.2) were seen in two cases each, and have never before been reported in haematological malignancy. All but four patients were treated on front‐line Cancer and Leukaemia Group B clinical protocols. The presence of −7 as a sole secondary abnormality was associated with a lower complete remission (CR) rate (P = 0·004), while the presence of ≥3 aberrations was associated with a higher CR rate (P = 0·009) and +der(22)t(9;22) with a higher cumulative incidence of relapse (P = 0·02). It will be of interest to see if newly diagnosed t(9;22)‐positive adult ALL patients with these and other secondary aberrations respond differently to treatment regimens that include imatinib mesylate.
Immunoglobulin heavy chain genes somatic hypermutations and chromosome 11q22‐23 deletion in classic mantle cell lymphoma: a study of the Swiss Group for Clinical Cancer Research
Authors: Bertoni Francesco , Conconi Annarita , Cogliatti Sergio B. , Schmitz Shu‐Fang Hsu , Ghielmini Michele , Cerny Thomas , Fey Martin , Pichert Gabriella , Bertolini Francesco , Ponzoni Maurilio , Baldini Luca , Jones Chris , Auer Rebecca , Zucca Emanuele , Cavalli Franco , Cotter Finbarr E. ,   for the Swiss Group for Clinical Cancer Research
Published: January 2004

Mantle cell lymphoma (MCL) shares immunophenotypic and karyotypic features with chronic lymphocytic leukaemia. The latter comprises two distinct entities with prognosis dependent upon immunoglobulin heavy chain (IgH) gene mutational status and the presence of 11q deletion. We evaluated the relevance of IgH gene mutational status, IgV gene family usage and presence of 11q deletion in a series of 42 histologically reviewed classical MCL cases to determine the prognostic impact. VH3 was the most common VH family, with VH3‐21 being the most frequent individual VH gene. Approximately 30% of the cases had a IgH somatic mutation rate higher than 2%, but was only higher than 4% in <10% of cases. Half of the cases had deletion of chromosome 11q21‐telomere (11q21‐>ter), with two minimal deleted regions, at 11q22.2 and 11q23.2. There was no association between 11q loss and IgH gene somatic mutation rate; the use of VH3‐21 gene could be associated with a better prognosis.
Increased CD11b neutrophil expression in Budd–Chiari syndrome or portal vein thrombosis secondary to polycythaemia vera
Authors: Alvarez‐Larrán Alberto , García‐Pagán Juan Carlos , Abraldes Juan G. , Arellano Eduardo , Reverter Juan Carlos , Bosch Jaume , Cervantes Francisco
Published: January 2004

Budd–Chiari syndrome and portal vein thrombosis (BCS/PVT) are frequently associated with polycythaemia vera (PV). In an attempt to elucidate the mechanisms of BCS/PVT secondary to PV (T‐PV), CD11b neutrophil expression, neutrophil oxidative burst and platelet–neutrophil complexes (PNC) were assessed in 17 such patients. Three groups served as controls: BCS/PVT not secondary to PV (T‐nPV; n = 20), PV without thrombosis (PV‐nT; n = 16), and healthy controls (HC; n = 20). Baseline CD11b expression (in mean fluorescence intensity units) was 101 [95% confidence interval (CI): 79–128] in T‐PV patients, versus 25 (95% CI: 18–35) in T‐nPV, 59 (95% CI: 43–80) in PV‐nT, and 34 (95% CI: 25–48) in HC (P < 0·001). After N‐formyl‐L‐methionyl‐L‐leucyl‐L‐phenylalanine activation, T‐PV patients also showed higher CD11b values: 190 (95% CI: 151–238), versus 55 (95% CI: 41–72) in T‐nPV, 111 (95% CI: 81–153) in PV‐nT, and 77 (95% CI: 63–95) in HC (P < 0·001). In BCS/PVT, CD11b neutrophil expression had 90% specificity and 100% sensitivity for the association with PV. Finally, PV patients had higher oxidative burst and PNC than T‐nPV patients or HC (P < 0·05). These results support a role for neutrophils in BCS/PVT secondary to PV and indicate that neutrophil CD11b expression could be of use for PV screening in BCS/PVT patients.
Standardization of the endogenous thrombin potential measurement: how to minimize the effect of residual platelets in stored plasma
Authors: Chantarangkul Veena , Clerici Marigrazia , Bressi Caterina , Tripodi Armando
Published: January 2004

Platelet contamination in stored plasma may affect coagulation assays, including the endogenous thrombin potential (ETP), which has been proposed for the investigation of hyper‐ and hypo‐coagulability. The current recommendation of filtering plasma before freezing cannot be always met. This study provides evidence that filtering frozen plasma after thawing, prior to testing, may help to eliminate the unwanted effect of residual platelets on the ETP. This may have important implications in future studies, as the ETP could be determined with plasma that have been collected without precautions relating to platelet contamination, as is the case for plasmas collected in epidemiological studies.
Magnetic resonance screening of iron status in transfusion‐dependent β‐thalassaemia patients
Authors: Ooi G. C. , Khong P. L. , Chan G. C. F. , Chan K. N. , Chan K. L. , Lam W. , Ng I. , Ha S. Y.
Published: January 2004

The clinical utility of dual sequence (T1‐ and T2‐weighted) magnetic resonance (MR) imaging in estimating liver iron concentration (LIC) in 32 transfusion‐dependent β‐thalassaemia major (24 females; age 18·5±5·9 years) patients on desferrioxamine was evaluated. Signal intensity ratios (SIR) between liver, spleen and pancreas to psoas muscle were determined on both sequences. Relationships between clinical and MR parameters, and accuracy of SIR thresholds in determining adequacy of chelation from LIC were analysed. Liver T1‐ and T2‐SIR were related to LIC (P < 0·001). T1‐SIR < 0·60 predicted severe iron overload (LIC > 15 mg/g) with sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) of 100%, 87%, 33% and 100% respectively. T2‐SIR < 0·1 yielded 100% sensitivity, 93% specificity, 50% PPV and 100% NPV. T1‐SIR ≥ 1·1 predicted LIC < 7 mg/g with 69% sensitivity, 88% specificity, 85% PPV and 74% NPV. T2‐SIR ≥ 0·20 yielded 56·5% sensitivity, 94% specificity, 90% PPV and 71% NPV. LIC correlated with liver T1‐SIR, liver T2‐SIR and serum ferritin (r = −0·76, −0·65, 0·47, respectively; P < 0·01). Serum ferritin was inversely related to liver T1‐SIR, liver T2‐SIR and spleen T2‐SIR (r = −0·35, −0·43, −0·40, respectively; P < 0·05). Mean total transfusion burden was not related to any MR parameter. Although neither MR sequence was a highly accurate predictor of LIC, SIR thresholds are useful to determine presence of iron overload and adequacy of chelation treatment.
Platelet microparticles induce angiogenesis in vitro
Authors: Kim Hyun Kyung , Song Kyung Soon , Chung Jun‐Ho , Lee Kyoung Rhan , Lee Se‐Na
Published: January 2004

Platelet microparticles (PMP) are endogenous substances generated during the coagulation process in a hypercoagulable state. This study demonstrated that PMP promote the proliferation and survival, migration, and tube formation in human umbilical vein endothelial cells (HUVEC). Heat‐treated PMP did not significantly decrease the angiogenic activity in HUVEC compared with that of the untreated PMP. Meanwhile when PMP were treated with activated charcoal, a procedure known to remove the lipid growth factors, the angiogenic activity was significantly reduced. These results suggest that the lipid component(s) of the PMP may be major active factor(s) and that protein component(s) may be minor contributor(s). PMP were also shown to augment endothelial progenitor cell differentiation in peripheral blood mononuclear cells. In addition, PMP‐stimulated proliferation, chemotaxis and tube formation of the HUVEC was mediated via the Pertussis toxin‐sensitive G protein, extracellular signal‐regulated kinase and the phosphoinositide 3‐kinase pathway. Herein, a new action of PMP was demonstrated to be a potent angiogenic stimulator. It is expected that in pathological states such as a growing tumour, PMP shed from the circulating platelets may reach adequate concentrations and that the elevated levels of PMP could contribute to florid formation of new blood vessels.
Genetic variability of von Willebrand factor and risk of coronary heart disease: the Rotterdam Study
Authors: Van Der Meer Irene M. , Brouwers Geert‐Jan , Bulk Saskia , Leebeek Frank W. G. , Van Der Kuip Deirdre A. M. , Hofman Albert , Witteman Jacqueline C. M. , Gómez García Encarnación B.
Published: January 2004

The von Willebrand factor (VWF) may be causally associated with coronary heart disease (CHD) or merely be a marker of endothelial damage. The G allele of the −1793 C/G promoter polymorphism in the VWF gene has been associated with higher plasma levels of VWF. To investigate whether VWF has a causal role in CHD, we designed a case‐cohort study, including 352 subjects with CHD and a random cohort (n = 736), and prospectively examined the association of the −1793 C/G polymorphism with CHD in subjects with and without advanced atherosclerosis. All subjects were ≤75 years of age and participating in the population‐based Rotterdam Study. Atherosclerosis was assessed by the ankle–arm index. Among subjects with advanced atherosclerosis, heterozygous and homozygous carriers of the G allele had a 3·5 (1·2–10·2) and 1·5 (0·4–5·7) fold increased risk of CHD respectively, compared with C/C homozygotes. The hazard ratio was 2·6 (1·0–6·8) for carriers of at least one copy of the G allele versus non‐carriers. No associations were found in the absence of advanced atherosclerosis. In conclusion, this study suggests that the G allele of the −1793 C/G polymorphism in the VWF gene is associated with an increased risk of CHD, but only in subjects with advanced atherosclerosis.
The effects of selective cytokine inhibitory drugs (CC‐10004 and CC‐1088) on VEGF and IL‐6 expression and apoptosis in myeloma and endothelial cell co‐cultures
Authors: Molostvov G. , Morris A. , Rose P. , Basu S. , Muller G.
Published: January 2004

Myeloma cells and human umbilical vein endothelial cells (HUVECs) were co‐cultured to model in vitro the interactions between myeloma and endothelium, and treated with thalidomide and two selective cytokine inhibitory drugs (SelCIDs, phosphodiesterase‐4 inhibitors). Flow cytometry and enzyme‐linked immunosorbent assay were used to assess production of two key cytokines – vascular endothelial growth factor (VEGF) and interleukin‐6 (IL‐6) – and apoptosis in co‐cultured HUVECs and myeloma cells. VEGF was produced by both myeloma cells and HUVECs, while IL‐6 was almost exclusively produced by endothelial cells. In co‐culture, there was significant up‐regulation of VEGF and IL‐6 production compared with the sum of separate myeloma and endothelial cell cultures. SelCIDs markedly inhibited production of both cytokines in co‐cultures, with CC‐10004 being more potent than CC‐1088. In addition, SelCIDs induced myeloma cell apoptosis. Apoptosis in co‐cultured myeloma cells was significantly lower than in those cultured separately, suggesting that co‐culture partially protected myeloma cells from drug‐induced apoptosis. This protective effect was probably due to IL‐6 produced by endothelial cells in co‐culture as addition of anti‐IL‐6 neutralizing antibody, but not anti‐VEGF antibody, abrogated it. In conclusion, SelCIDs can exert their anti‐myeloma activity through two mechanisms, i.e. inhibition of VEGF and IL‐6 production by interacting myeloma and endothelium and induction of myeloma cell apoptosis.
A patient homozygous for a Gly354Cys mutation in factor VII that results in severely impaired secretion of the molecule, but not complete deficiency
Authors: Takamiya Osamu , Hino Kenichiro
Published: January 2004

We investigated the molecular basis of factor VII (FVII) deficiency in a Japanese woman who suffered occasional epistaxis. The patient had low levels of both FVII coagulant activity (FVII:C) and antigen (FVII:Ag) (5·0% and 7·3% of normal controls respectively). DNA sequence analysis of the FVII gene showed that the patient was homozygous for a mutation that resulted in a Cys for Gly354 substitution, a novel missense mutation in the catalytic domain. Haplotype analysis showed that this missense mutation was inherited from her consanguineous parents. Transient expression experiments showed that secreted FVII Cys354, FVII:C and FVII:Ag levels in conditioned media were reduced to 4% and 5%, respectively, of levels secreted from wild‐type FVII. However, the intracellular FVII Cys354 was 67% that of normal recombinant protein. Immunohistochemical analysis showed that intracellular FVII:Ag from FVII 354Cys was present diffusely throughout the cytoplasm. Substitution of FVII 354Gly with amino acids other than Cys (Arg, Asp, Ser and Phe), did not produce a phenotype similar to FVII Cys354Gly. Molecular modelling indicated that FVII Gly354 was located outside the FVII heavy chain, and that Cys135 in the EGF2 domain, Cys262 in the catalytic domain and Cys127 all exist within 10 Å of Gly354. Therefore, we propose that the introduction of an additional free cysteine residue in the FVII molecule results in the formation of illegitimate disulphide bonds and a mis‐folded domain, leading to defective secretion.
Systemic AL amyloidosis due to non‐Hodgkin's lymphoma: an unusual clinicopathologic association
Authors: Cohen A. D. , Zhou P. , Xiao Q. , Fleisher M. , Kalakonda N. , Akhurst T. , Chitale D. A. , Moscowitz C. , Dhodapkar M. V. , Teruya‐Feldstein J. , Filippa D. , Comenzo R. L.
Published: January 2004

Systemic AL amyloidosis (AL) is a disorder in which light chains form fibrillar deposits, leading to organ dysfunction and death. Rarely, AL has been associated with non‐Hodgkin's lymphoma (NHL), although this association has not been well characterized. We report a series of six patients with AL associated with NHL, primarily lymphoplasmacytic lymphoma. Organ involvement was variable, with frequent bulky lymphadenopathy and visceral cavity deposits, but no cardiac involvement. Positron emission tomography scans were negative. Bone marrow and lymph node biopsies showed a mixed population of CD20+ lymphoid and CD138+ plasma cells. Serum free light chains were elevated, and correlated with response to therapy. Immunoglobulin light chain variable region (Ig VL) germline gene use was typical for AL, reflecting previously observed correlations between germline gene use and organ tropism. Five patients received rituximab‐based therapies with two responses. Two patients underwent autologous stem cell transplantation with one complete haematological response. Four patients survive at 10–132 months from diagnosis. AL with NHL has distinctive clinical features but employs the same Ig VL gene repertoire as AL with clonal plasma cell dyscrasias. Serial serum free light chain levels are useful for tracking response to therapy. Treatments aimed at both lymphoid and plasma cell components appear warranted.
Cdk‐inhibitors and exit from quiescence in primitive haematopoietic cell subsets
Authors: Steinman Richard , Yaroslavskiy Beatrice , Goff Julie P. , Alber Sean M. , Watkins Simon C.
Published: January 2004

Prolonged quiescence of haematopoietic stem cells has been proposed to support durable haematopoiesis through clonal succession. Genetic experiments in mice have implicated the cyclin‐dependent kinase inhibitor (cdki) p21Waf1 in sustaining stem cell quiescence, and the cdki p27Kip1 in inhibiting the expansion of more mature progenitor cells. The expression of these inhibitory proteins in human haematopoietic stem cell candidates has not hitherto been studied. We describe a rare subpopulation (3 × 10−7 umbilical cord mononuclear cells) of lineage‐negative cells that exhibited sustained resistance over months to cytokine‐induced cycling, and characterized the expression of p21Waf1 and p27Kip1 proteins in these cells. Whereas p27Kip1 was uniformly expressed in these cells, the expression of p21Waf1 in this population and in lineage‐negative CD34+ cells was variable. For this rare subset of cells exhibiting prolonged quiescence, p21Waf1 may be dispensable and p27Kip1 necessary for growth arrest.
Reactive oxygen species and phosphatidylserine externalization in murine sickle red cells
Authors: Banerjee Tinku , Kuypers Frans A.
Published: January 2004

Due to their role in oxygen transport and the presence of redox active haemoglobin molecules, red blood cells (RBC) generate relatively high levels of reactive oxygen species (ROS). To counteract the potential deleterious effects of ROS, RBCs have a well‐integrated network of anti‐oxidant mechanisms to combat this oxidative stress. ROS formation is increased in sickle‐cell disease (SCD) and our studies in a murine SCD model showed a significant increase in the generation of ROS when compared with normal mice. Our data also indicated that murine sickle RBCs exhibit a significantly increased ATP catabolism, partly due to the increased activity of glucose‐6‐phosphate dehydrogenase and glutathione reductase to regenerate intracellular glutathione (GSH) levels to neutralize the adverse milieu of oxidative stress. Higher ATP consumption by the murine sickle RBCs, together with the increased ROS formation and impairment of the aminophospholipid translocase or flipase may underlie the exposure of phosphatidylserine on the surface of these cells.
CD27‐triggering on primary plasma cell leukaemia cells has anti‐apoptotic effects involving mitogen activated protein kinases
Authors: Guikema Jeroen E. J. , Vellenga Edo , Abdulahad Wayel H. , Hovenga Sjoerd , Bos Nicolaas A.
Published: January 2004

Primary plasma cell leukaemia (PCL) is a rare plasma cell malignancy, which is related to multiple myeloma (MM) and is characterized by a poor prognosis. In a previous study we demonstrated that PCL plasma cells display a high expression of CD27, in contrast to MM plasma cells. The present study was set out to assess the functional properties of CD27 expressed on PCL plasma cells by triggering with its ligand CD70. Using CD27‐expressing purified plasma cells from a PCL patient we demonstrated that CD27‐triggering modestly inhibited spontaneous and dexamethasone‐induced apoptosis. In vitro stimulation and Western blotting showed that activation of p38 and extracellular‐regulated kinase 1/2 (ERK1/2) mitogen‐activated protein kinases (MAPK) was associated with CD27‐mediated signal transduction. Specific inhibition of p38 and ERK1/2 MAPK abolished the anti‐apoptotic effects of CD27‐triggering. Interestingly, simultaneous inhibition of p38 and ERK1/2 strongly sensitized PCL cells for dexamethasone‐induced apoptosis. Finally, in dexamethasone‐treated PCL cells, CD27‐triggering was associated with persistent DNA‐binding activity of activator protein 1 (AP‐1) but not of nuclear factor‐κB. These findings suggest that, in primary PCL, specific anti‐apoptotic pathways exist that might provide novel therapeutic targets.
Dietary vitamin K influences intra‐individual variability in anticoagulant response to warfarin
Authors: Khan T. , Wynne H. , Wood P. , Torrance A. , Hankey C. , Avery P. , Kesteven P. , Kamali F.
Published: January 2004

The relationship between dietary intake of vitamin K, fat, plasma vitamin K concentrations and anticoagulation response to warfarin within individuals, as well as the contribution of dietary vitamin K to differences in warfarin dose requirements between individuals were investigated in 53 patients on warfarin therapy who had stably controlled anticoagulation. Each patient completed a dietary record of all foods consumed on a daily basis for 4 weeks. Each week a blood sample was taken for measurement of the international normalized ratio (INR), plasma vitamin K, triglycerides and warfarin enantiomer concentrations. The patients’ genotype for CYP2C9 was also determined. Regression analysis of the data showed that, for each increase of 100 μg in the daily dietary intake of vitamin K averaged over 4 d, the INR was reduced by 0·2. There was no correlation between warfarin daily dose and average daily dietary vitamin K intake when calculated over 28 d. The regression model for warfarin dose showed that, while dietary vitamin K had no effect, CYP2C9 genotype (P = 2%) and age (P < 1%) significantly contributed to inter‐patient variability in warfarin dose requirements. A consistent intake of vitamin K could reduce intrapatient variability in anticoagulation response and thus improve the safety of warfarin therapy.
Signalling molecules and cytokine production in T cells of multiple myeloma‐increased abnormalities with advancing stage
Authors: Mozaffari Fariba , Hansson Lotta , Kiaii Shahryar , Ju Xiuli , Rossmann Eva D. , Rabbani Hodjattallah , Mellstedt Håkan , Österborg Anders
Published: January 2004

T‐cell immune dysfunction in patients with malignant tumours has been attributed to the altered expression of components of the T‐cell receptor (TCR)/CD3 complex and their associated intracellular protein tyrosine kinases. In this study, four‐colour flow cytometry was applied to study the surface bound molecules TCRαβ, CD28, CD152 and CD154 involved in T‐cell signalling and the signal transduction molecules CD3ζ, p56lck, p59fyn, ZAP‐70 and phosphatidyl‐inositol‐3 kinase (PI3‐k) as well as the intracellular cytokines interferon‐γ (IFN‐γ), interleukin (IL)‐4 and IL‐2 as a functional read‐out of non‐stimulated and superantigen (staphylococcus enterotoxin B)‐stimulated blood T cells of multiple myeloma (MM) patients at different stages of the disease. Multiple abnormalities were demonstrated in the CD4 and CD8 populations, both under non‐stimulated and superantigen‐stimulated conditions. There was a marked reduction, particular in advanced stage MM, in the proportion of CD4 and CD8 cells expressing CD28, CD152, CD3ζ, p56lck, ZAP‐70 and PI3‐k. The level of intracellular T‐cell cytokines (IFN‐γ, IL‐2 and IL‐4) was normal or increased in non‐stimulated cells but activation‐induced cytokine production was impaired. These results illustrated profound and multiple T‐cell signalling defects, from the surface and down‐stream, consistent with involvement of a master T‐cell function, especially in advanced stage MM. These data should be taken into consideration when developing immune‐based therapeutic approaches and when applying new emerging technologies that aim to restore T‐cell functions.
Reliable detection of clonal IgH/Bcl2 MBR rearrangement in follicular lymphoma: methodology and clinical significance
Authors: Iqbal Sameena , Jenner Michael J. R. , Summers Karin E. , Davies Andrew J. , Matthews Janet , Norton Andrew J. , Calaminici Maria , Rohatiner Ama Z. , Fitzgibbon Jude , Lister T. Andrew , Goff Lindsey K.
Published: January 2004

The prognostic significance of IgH/Bcl2 rearrangement in follicular lymphoma (FL) remains contentious; polymerase chain reaction (PCR) methodology and tissue source variability may account for some inconsistencies. As IgH/Bcl2 major breakpoint region (MBR) sequences may be found in normal blood, an MBR+ result by conventional PCR in blood/bone marrow may not indicate FL. To establish tumour MBR status, 190 lymphoid tissue samples with histologically evident FL (and therefore >1% tumour cells) were examined by real‐time quantifiable PCR; 50% (95/190) had clonal MBR IgH/Bcl2 (MBR was considered clonal when >1%). Overall survival (median = 11·5 years) of MBR+ and MBR− patients was not significantly different.
Monitoring the development of clinical skills during training in a clinical placement
Authors: Pender F. T. , de Looy A. E.
Published: January 2004

Background  The education and training of health care practitioners has undergone recent reform, and indicate that curricula should place emphasis on the development of clinical skills and attitude. The purpose of this study was two‐fold: to define the key skills necessary for a competent dietetic student practitioner and to devise a reliable assessment tool to measure and track performance in these key skill areas throughout the period of clinical placement.

Methods  Key clinical skills were identified by a concensus group of experienced dietitians and academic practitioners. An assessment tool was then developed to measure these attributes in 43 students undertaking clinical placement at a number of training centres in Scotland. Development of skills was tracked for the entire duration of placements using the novel assessment tool. The assessment tool used a visual analogue scale (VAS) as the measuring score.

Results  A high level of skill attainment was equated with high VAS scores. Performance in three of the four key skills (written skills: r = 0.762, P ≤ 0.001; interviewing skills: r = 0.697, P < 0.001; and dietary assessment technique: r = 0.697, P ≤ 0.001) showed impressive correlation co‐efficients, indicating a striking and significant positive correlation with the length of training. A high skill performance level attainment was achieved by week 16/17 of training.

Conclusion  The authors are confident that the assessment tool is valid and reliable and measures skill performance objectively. A high level of skill attainment was observed in most students by about the mid‐point in training: this allows the student to consolidate these skills in the latter stages of training which is in keeping with educational and practice philosophy of placement learning outcomes.