Alpha Fragment Research Articles

Characteristics of alpha projectile fragments emission in interaction of nuclei with emulsion

The properties of the relativistic alpha fragments produced in interactions of 84^Kr at around 1 A GeV in nuclear emulsion are investigated. The experimental results are compared with the similar results obtained from various projectiles with emulsion interactions at different energies. The total, partial nuclear cross-sections and production rates of alpha fragmentation channels in relativistic nucleus-nucleus collisions and their dependence on the mass number and initial energy of the incident projectile nucleus are investigated. The yields of multiple alpha fragments emitted from the interactions of projectile nuclei with the nuclei of light, medium and heavy target groups of emulsion-detector are discussed and they indicate that the projectile-breakup mechanism seems to be free from the target mass number. It is found that the multiplicity distributions of alpha fragments are well described by the Koba-Nielsen-Olesen (KNO) scaling presentation. The mean multiplicities of the freshly produced newly created charged secondary particles, normally known as shower and secondary particles associated with target in the events where the emission of alpha fragments were accompanied by heavy projectile fragments having Z value larger than 4 seem to be constant as the alpha fragments multiplicity increases, and exhibit a behavior independent of the alpha fragments multiplicity.

Immunohistochemical detection of prothymosin alpha in pituitary adenomas--a new marker of tumor recurrence?

Forty pituitary adenomas were immunostained with an antibody raised against the C-terminal fragment (101-109) of human prothymosin alpha (PT alpha). The strong positive immunostaining was found in the subpopulation of cell nuclei and intratumoral vessel walls, while the cytoplasm of adenoma cells was slightly immunopositive. The significantly higher percentage of PT alpha-positive cell nuclei was found in recurrent pituitary adenomas as compared with primary tumors. However, there was no correlation between the percentage of PT alpha-positive cell nuclei and Ki-67 indices. Gonadotropinomas were characterized by higher nuclear PT alpha expression in comparison to other pituitary adenomas, which is probably linked with the high recurrence rate of these tumors. It is suggested that PT alpha immunostaining may be helpful in predicting the pituitary tumor recurrence. However, this conclusion needs to be confirmed in further prospective studies. Moreover, PT alpha may be also useful as an immunohistochemical marker of the intratumoral microvasculature.

Cerebrospinal fluid biomarkers of white matter lesions – cross‐sectional results from the LADIS study

White matter lesions (WMLs) caused by small vessel disease are common in elderly people and contribute to cognitive impairment. There are no established biochemical markers for WMLs. We aimed to study the relation between degree of WMLs rated on magnetic resonance imaging of the brain and cerebrospinal fluid (CSF) levels of structural biomarkers associated with Alzheimer's disease (AD) and subcortical vascular dementia. Fifty-three non-demented elderly individuals with WMLs were subjected to lumbar puncture. Degree of WMLs was rated using the Fazekas scale. Volumetric assessment of WMLs was performed. CSF samples were analyzed for the 40 and 42 amino acid fragments of amyloid beta, alpha- and beta-cleaved soluble amyloid precursor protein, total tau (T-tau), hyperphosphorylated tau (P-tau(181)), neurofilament light protein (NFL), sulfatide and CSF/Serum-albumin ratio. Fifteen subjects had mild, 23 had moderate and 15 had severe degree of WMLs. CSF-NFL levels differed between the groups (P < 0.001) and correlated with the volume of WMLs (r = 0.477, P < 0.001). CSF sulfatide concentration displayed similar changes but less strongly. T-tau, P-tau(181) and the different amyloid markers as well as CSF/S-albumin ratio did not differ significantly between the groups. The association of increased CSF-NFL levels with increasing severity of WMLs in non-demented subjects suggests that NFL is a marker for axonal damage in response to small vessel disease in the brain. This manifestation may be distinct from or earlier than the neurodegenerative process seen in AD, as reflected by the lack of association between WMLs and AD biomarkers.

Staphylococcal superantigen-like 5 activates platelets and supports platelet adhesion under flow conditions, which involves glycoprotein Ibalpha and alpha IIb beta 3.

Staphylococcal superantigen-like 5 (SSL5) is an exoprotein secreted by Staphylococcus aureus that has been shown to inhibit neutrophil rolling over activated endothelial cells via a direct interaction with P-selectin glycoprotein ligand 1 (PSGL-1). When purified recombinant SSL5 was added to washed platelets in an aggregometry set-up, complete and irreversible aggregation was observed. Proteolysis of the extracellular part of GPIb alpha or the addition of dRGDW abrogated platelet aggregation. When a mixture of isolated platelets and red cells was perfused over immobilized SSL5 at a shear rate of 300 s(-1), stable platelet aggregates were observed, and platelet deposition was substantially reduced after proteolysis of GPIb or after addition of dRGDW. SSL5 was shown to interact with glycocalicin, a soluble GPIb alpha fragment, and binding of SSL5 to platelets resulted in GPIb-mediated signal transduction as evidenced by translocation of 14-3-3 zeta. In addition, SSL5 was shown to interact with endothelial cell matrix (ECM) and this interaction enhanced aggregation of platelets from whole blood to this ECM. SSL5 activates and aggregates platelets in a GPIb alpha-dependent manner, which could be important in colonization of the vascular bed and evasion of the immune system by S. aureus.

Purification and characterization of a transgenic corn grain‐derived recombinant collagen type I alpha 1

Corn offers advantages as a transgenic host for producing recombinant proteins required at large volumes (1,000's of tons per year) and low cost (less than US$50/kg) by generating them as co-products of biorefining. We describe the purification and characterization of a corn grain-derived mammalian structural protein having such market characteristics: a full length recombinant collagen type I alpha 1 (rCI alpha 1) chain. Material properties of interest are gelation behavior, which would depend on as yet unverified ability of corn to carry out post-translational prolyl hydroxylation and formation of triple helical conformation. The starting material was grain where the expression of rCI alpha 1 had been directed by an embryo-specific promoter. Purification consisted of extraction at low pH followed by membrane and chromatographic steps to isolate rCI alpha 1 for characterization. The amino acid composition and immunoreactivity of CI alpha 1 was similar to that of an analogous native human CI alpha 1 and to rCI alpha 1 produced by the yeast Pichia pastoris. Tandem mass spectrometry confirmed the primary sequence of the corn-derived rCI alpha 1 with 46% coverage. Fragments of the rCI alpha 1 chains were also observed, possibly caused by endogenous plant proteases. The corn-derived rCI alpha 1 had a low level of prolyl hydroxylation (approximately 1% versus 11%) relative to animal-derived CI alpha 1 and folded into its characteristic triple-helical structure as indicated by its resistance to pepsin digestion below its melting temperature of 26(o)C. The 29 amino acid foldon fused to the C-terminus to initiate triple helix formation was not cleaved from the rCI alpha 1 chains, but could be removed by pepsin treatment.

Prion protein region 23–32 interacts with tubulin and inhibits microtubule assembly

In previous studies we have demonstrated that prion protein (PrP) binds directly to tubulin and this interaction leads to the inhibition of microtubule formation by inducement of tubulin oligomerization. This report is aimed at mapping the regions of PrP and tubulin involved in the interaction and identification of PrP domains responsible for tubulin oligomerization. Preliminary studies focused our attention to the N-terminal flexible part of PrP encompassing residues 23-110. Using a panel of deletion mutants of PrP, we identified two microtubule-binding motifs at both ends of this part of the molecule. We found that residues 23-32 constitute a major site of interaction, whereas residues 101-110 represent a weak binding site. The crucial role of the 23-32 sequence in the interaction with tubulin was confirmed employing chymotryptic fragments of PrP. Surprisingly, the octarepeat region linking the above motifs plays only a supporting role in the interaction. The binding of Cu(2+) to PrP did not affect the interaction. We also demonstrate that PrP deletion mutants lacking residues 23-32 exhibit very low efficiency in the inducement of tubulin oligomerization. Moreover, a synthetic peptide corresponding to this sequence, but not that identical with fragment 101-110, mimics the effects of the full-length protein on tubulin oligomerization and microtubule assembly. At the cellular level, peptide composed of the PrP motive 23-30 and signal sequence (1-22) disrupted the microtubular cytoskeleton. Using tryptic and chymotryptic fragments of alpha- and beta-tubulin, we mapped the docking sites for PrP within the C-terminal domains constituting the outer surface of microtubule.

α- and β-tubulin from Phytophthora capsici KACC 40483: molecular cloning, biochemical characterization, and antimicrotubule screening

Internal fragments of alpha- and beta-tubulin genes were generated using reverse transcription polymerase chain reaction (RT-PCR), and the termini were isolated using 5'- and 3'-rapid amplification of cDNA ends. Phytophthora capsici alpha- and beta-tubulin specific primers were then used to generate full-length cDNA by RT-PCR. The recombinant alpha- and beta-tubulin genes were expressed in Escherichia coli BL21 (DE3), purified under denaturing conditions, and average yields were 3.38-4.5 mg of alpha-tubulin and 2.89-4.0 mg of beta-tubulin, each from 1-l culture. Optimum conditions were obtained for formation of microtubule-like structures. A value of 0.12 mg/ml was obtained as the critical concentration of polymerization of P. capsici tubulin. Benomyl inhibited polymerization with half-maximal inhibition (IC(50)) = 468 +/- 20 microM. Approximately 18.66 +/- 0.13 cysteine residues per tubulin dimer were accessible to 5,5'-dithiobis-(2-nitrobenzoic acid), a quantification reagent of sulfhydryl and 12.43 +/- 0.12 residues were accessible in the presence of 200 microM benomyl. The order of preference for accessibility to cysteines was benomyl > colchicine > GTP > taxol, and cysteine accessibility changes conformed that binding sites of these ligands in tubulin were folding correctly. Fluorescence resonance energy transfer technique was used for high throughput screening of chemical library in search of antimitotic agent. There was significant difference in relative fluorescence by 210-O-2 and 210-O-14 as compared to colchicine.

Three new potential ovarian cancer biomarkers detected in human urine with equalizer bead technology

To examine whether urine can be used to measure specific ovarian cancer proteomic profiles and whether one peak alone or in combination with other peaks or CA125 has the sensitivity and specificity to discriminate between ovarian cancer pelvic mass and benign pelvic mass. A total of 209 women were admitted for surgery for pelvic mass at the Gynaecological Department at Rigshospitalet, Copenhagen. Of the women, 156 had benign gynaecological tumors, 13 had borderline tumors and 40 had malignant epithelial ovarian cancer. The prospectively and preoperatively collected urine samples were aliquotted and frozen at -80 degrees until the time of analysis. The urine was fractionated using equalizer bead technology and then analyzed with surface-enhanced laser desorption/ionization time-of-flight mass spectrometry. Biomarkers were purified and identified using combinations of chromatographic techniques and tandem mass spectrometry. Benign and malignant ovarian cancer cases were compared; 21 significantly different peaks (p<0.001) were visualized using Mann-Whitney analysis, ranging in m/z values from 1,500 to 185,000. The three most significant peaks were purified and identified as fibrinogen alpha fragment (m/z=2570.21), collagen alpha 1 (III) fragment (m/z=2707.32) and fibrinogen beta NT fragment (m/z=4425.09). The area under the receiver operator characteristic curve (ROC AUC) value for these three peaks in combination was 0.88, and their ROC AUC value in combination with CA125 was 0.96. This result supports the feasibility of using urine as a clinical diagnostic medium, and the ROC AUC value for the three most significant peaks in combination with or without CA125 demonstrates the enhanced prediction performance of combined marker analysis.

Genotyping of Clostridium perfringens isolated from calves with neonatal diarrhea

Neonatal diarrhea is one of the main causes of losses in cattle herds. Clostridium perfringens is a widespread enteropathogen, and is responsible for many animal diseases such as bovine neonatal diarrhea. Fecal samples from 141 diarrheic calves and 129 healthy calves, aged up to 28 days and belonging to three herds were examined. Rates of culture positivity were 36.2% and 30.2% for diarrheic and nondiarrheic calves, respectively. Multiple isolates from primary isolation plates were subjected to simultaneous genotyping by multiplex PCR, with primers amplifying fragments of alpha ( cpa), beta ( cpb), epsilon ( etx), iota ( itxA), enterotoxin ( cpe) and beta2 ( cpb2) toxin-encoding genes. Only 17/51 (33.3%) and 17/39 (43.6%) of these mixtures from diarrheic and nondiarrheic calves, respectively, yielded genotype information, suggesting that this may not be a viable approach to genotyping of isolates. Fourteen isolate mixtures from animals with diarrhea had only cpa (type A), one had cpa and cpb2 (type A beta2 positive), one with cpa, itxA, and cpb2 (type E, beta2 positive), and one with cpa, etx, itxA, and cpb2 toxin producing strains. Among 17 isolate mixtures from healthy calves, 10 were exclusively type A, one was type A cpb2 positive, two were type E, three were type E cpb2 positive, and one was types D and E cpb2 positive. There was no correlation between isolation of a given toxin type and the presence of diarrhea.

Effect of overexpression of inhibin α (1–32) fragment on bovine granulosa cell proliferation, apoptosis, steroidogenesis, and development of co-cultured oocytes

The objective was to determine the effects of an inhibin alpha (1-32) fragment gene on proliferation, apoptosis, and steroidogenesis of bovine granulosa cells (GC) isolated from medium and small follicles (diameter >4-8 and 1-4mm, respectively), and the effect of GC, previously transfected with pEGISI, on oocyte maturation and in vitro embryo development. To enhance expression of the inhibin alpha (1-32) fragment, GC were transfected with pEGISI. Transfection inhibited (P<0.05) GC proliferation (88.8+/-2.1%; mean+/-S.E.M.) compared to the control and EGFP groups (100% and 97.5+/-2.1%) from medium follicles, with no significant effect on GC from small follicles. Apoptosis was higher (P<0.01) in transfected GC than in controls. Transfection increased (P<0.05) estradiol synthesis from both medium and small follicles (0.57+/-0.13 and 0.86+/-0.13 pg/mL vs. 0.19+/-0.05 and 0.35+/-0.09 pg/mL in controls) after culturing for 48 h, with suppression (P<0.05) in transfected GC after 96 h. Transfection reduced (P<0.05) progesterone synthesis in GC from both medium and small follicles (24.5+/-3.4 and 75.4+/-4.6 ng/mL vs. 45.42+/-5.33 and 117.32+/-11.99 ng/mL in controls) after culture for 48 h, with no significant difference after 96 h. Maturation rate of oocytes co-cultured with transfected GC from medium follicles was decreased relative to control (61.5+/-6.8% vs. 71.2+/-5.7%, P<0.05), with no significant effect on embryo development. In conclusion, overexpression of inhibin alpha (1-32) fragment regulated GC development; effects on subsequent oocyte maturation were both time- and stage-dependent.

Integral frequency distribution of projectile alpha fragments in nuclear multifragmentations at intermediate and high energies

The integral frequency distributions of the numbers of projectile alpha fragments produced in nuclear multifragmentations at intermediate and high energies are studied by a simple two-source model. The calculated results are approximately in agreement with the experimental data of 16 O, 40 Ar, 56 Fe, 84 Kr, 139 La, and 197 Au multifragmentations in nuclear emulsions at 2.00 A , 1.80 A , 1.80 A , 0.08–1.70 A , 0.60–0.99 A , and 0.60–0.99 A GeV, respectively. The initial energy of nuclear limiting fragmentation is expected to be at or lower than 0.08 A GeV. Two correlations between the mean multiplicity of projectile alpha fragments ( 〈 n α 〉 ) and the projectile mass number, as well as 〈 n α 〉 and the maximum multiplicity of projectile alpha fragments are observed.

Specific MALDI Imaging and Profiling for Biomarker Hunting and Validation: Fragment of the 11S Proteasome Activator Complex, Reg Alpha Fragment, Is a New Potential Ovary Cancer Biomarker

MALDI imaging mass spectrometry represents a new analytical tool to directly provide the spatial distribution and relative abundance of proteins in tissue. Twenty-five ovary carcinomas (stages III and IV) and 23 benign ovaries were directly analyzed using MALDI-TOF MS. The biomarker with the major prevalence (80%) has been fully identified using MALDI MS and nanoESI MS and MS/MS after separation by RP-HPLC and trypsin enzymatic digestion. This marker with an m/z of 9744 corresponds to 84 amino acid residues from the 11S proteasome activator complex, named PA28 or Reg-alpha. Validation of this marker has been performed using MALDI imaging, classical immunocytochemistry with an antibody raised against the C-terminal part of the protein, specific MALDI imaging, and Western blot analysis. The validation, using immunocytochemistry, confirmed the epithelial localization of this fragment with nucleus localization in benign epithelial cells and a cytoplasmic localization in carcinoma cells. This indicates that this antibody could be used to discriminate the borderline tumor cases. At this point, a multicentric study needs to be conducted in order to clearly establish the potential of this biomarker. Taken together these studies reflect that direct tissue analysis and specific MALDI imaging strategies facilitate biomarker hunting and validation which can be named pathological proteomics.

Alpha melanocyte stimulating hormone and fragments: Components of the cutaneous defense and therapeutic potential

Alpha melanocyte stimulating hormone and fragments: Components of the cutaneous defense and therapeutic potential

Yield improvement for manufacture of α1‐proteinase inhibitor

The aim of this study was to increase the yield of active alpha(1)-proteinase inhibitor (alpha(1)-PI) from Cohn fraction IV-1 paste during the manufacture of this therapeutic protein and to investigate the molecular mechanism for this yield increase. Dissolution experiments with IV-1 paste investigated the impact of different variables on the yield of alpha(1)-PI activity. Solutions of IV-1 paste prepared under different conditions were assayed for evidence of protease activity by Western blots of alpha(1)-PI following SDS-PAGE, by azocaseinolytic and amidolytic (S-2288) assays, and by zymography, and for the extent of alpha(1)-PI oligomerization by Western blots following nondenaturing PAGE. Minor modification of the manufacturing process by combining dissolution of IV-1 paste with the subsequent pH adjustment (to 9.25-9.50 with NaOH), achieved by addition of a standard concentration of NaOH to the 10-mm Tris base dissolvent for IV-1 paste, was found to give a highly reproducible 9.4 +/- 0.9% increase in yield of active alpha(1)-PI. Solutions of IV-1 paste prepared with this combined dissolvent contained reduced amounts of low molecular weight fragments of alpha(1)-PI, reduced protease activity, and reduced amounts of oligomers of alpha(1)-PI. Addition of the protease inhibitor leupeptin to the 10-mm Tris base dissolvent for IV-1 paste also caused an increase in the yield of alpha(1)-PI activity. Dissolution of IV-1 paste in a more alkaline medium gave a significant increase in the yield of active alpha(1)-PI. This yield increase was attributed to a reduction both in protease activity and in the extent of oligomerization of alpha(1)-PI.

Declining Plasma Fibrinogen Alpha Fragment Identifies HER2-Positive Breast Cancer Patients and Reverts to Normal Levels after Surgery

Breast cancer is the most common nonskin malignancy affecting women. Currently, no simple, blood-based diagnostic test exists to complement radiological screening and increase sensitivity of detection. To screen plasma specimens and identify biomarkers that detect HER2-positive breast cancer, automated robotic sample processing followed by surface-enhanced laser desorption ionization time-of-flight (SELDI-TOF) mass spectroscopy was used. Multiple statistical algorithms were used to select biomarkers that segregate cancer patients versus controls and produced average CV rates ranging from 20% to 29%. A set of seven biomarkers were validated on an independent test data set and achieved the best error rate of 19.1%. A permutation test indicated a p-value for CV error less than 0.002. Moreover, a ROC curve using these biomarkers achieved an area-under-the-curve value of 0.95 on an independent test data set. The marker responsible for most of the resolving power was identified as a fragment of Fibrinogen Alpha (FGA) encompassing residues 605-629. This marker was present at lower levels in cancer patients as compared to controls. The importance of this biomarker was validated in a longitudinal study comparing pre- and post-operative levels and was shown to revert to normal levels after surgery. This fragment may serve as a useful diagnostic and treatment-monitoring marker.

A Homogeneous Cell-Based Assay to Measure Nuclear Translocation Using β-Galactosidase Enzyme Fragment Complementation

Positional complementation describes the use of homogeneous assays using beta- galactosidase (beta gal) enzyme fragment complementation to detect cellular protein translocation. This phenomenon occurs when the protein of interest, recombinantly expressed as a fusion protein with a modified alpha fragment of beta gal, translocates to a cellular compartment expressing an enzyme acceptor fragment of the enzyme. When these fragments interact, high-affinity complementation occurs, and a signal is generated that is then detected upon cell lysis. In the present paper the use of positional complementation is exemplified by measuring nuclear translocation of the glucocorticoid receptor in Chinese hamster ovary-K1 cells. The approach thus provides for homogeneous protocols, in an endpoint microtiter plate assay format, without the use of either imaging or reporter gene techniques. Consequently, these characteristics suggest that the technique is suitable for automated instrumentation protocols used in high throughput screening campaigns designed to identify activators or inhibitors of nuclear translocation.

Host Blood Proteins and Peptides in the Midgut of the Tick Dermacentor variabilis Contributeto Bacterial Control

Antimicrobial midgut proteins and peptides that result from blood digestion in feeding American dog ticks Dermacentor variabilis (Say) were identified. Midgut extracts from these ticks showed antimicrobial activity against Micrococcus luteus, regardless of whether they were challenged with peptidoglycan, blood meal components, rabbit blood, Bacillus subtilis, Escherischia coli or Borrelia burgdorferi. However, no peptide band co-migrating with defensin was found in midgut extracts from the challenged ticks. Partial purification of the midgut extracts using C(18) Sep Paks and gel electrophoresis showed the presence of 4 distinct bands with rMW 4.1, 5.3, 5.7 and 8.0 kDa identified by tryptic digestion-mass fingerprinting as digestive fragments of rabbit alpha-, beta-, gamma-chain hemoglobin, and rabbit ubiquitin. No evidence of varisin, a defensin previously identified in the hemolymph of D. variabilis, was found in the tryptic digest, although varisin was found in a hemocyte lysate using the same methods. However, varisin transcript was detected in midgut cell lysates. Also present in all midgut samples was a cluster of 3 overlapping bands with rMW 13.0, 14.1 and 14.7 kDa which were identified by tryptic-digestion LC-MS and MALDI-TOF as rabbit alpha- and beta-chain hemoglobin (undigested) and transtherytin. Lysozyme transcript was detected in midgut cell extracts but the peptide was not. Studies done on other tick species demonstrated that hemoglobin digestion resulted in antimicrobial fragments. Antimicrobial hemoglobin fragments (including fragments larger than any reported previously) also were found in D. variabilis, as well as ubiquitin, a peptide known to occur as part of an antimicrobial complex in vertebrate leukocytes. In addition, we noted that Borrelia burgdorferi spirochetes were not lysed in the midgut lumen, which would be expected if defensin and lysozyme were active in this location. In this respect, the midgut's response to microbial challenge differs from that of the hemolymph. In summary, the midgut's antimicrobial activity appears to be primarily a byproduct of hemoglobin digestion rather than expression of immune peptides and proteins.

Biologically active milli-calpain associated with caveolae is involved in a spatially compartmentalised signalling involving protein kinase C alpha and myristoylated alanine-rich C-kinase substrate (MARCKS)

We have previously shown that calpain promotes myoblast fusion by acting on protein kinase C-alpha and the cytosolic phosphorylated form of MARCKS. In other cell types, various isoforms of calpain, PKC alpha and MARCKS were found associated with caveolae. These vesicular invaginations of the plasma membrane are essential for myoblast fusion and differentiation. We have isolated caveolae from myoblasts and studied the presence of calpain isoforms and their possible effects on signalling mediated by caveolae-associated PKC. Our results show that milli-calpain co-localizes with myoblast caveolae. Futhermore we provide evidence, using a calcium ionophore and a specific inhibitor of calpains (calpastatin peptide), that milli-calpain reduces the PKC alpha and MARCKS content in these structures. Purified milli-calpain causes the appearance of the active catalytic fragment of PKC alpha (PKM), without having an effect on MARCKS. Addition of phorbol myristate acetate, an activator of PKC, induces tranlocation of PKC alpha towards caveolae and results in a significant reduction of MARCKS associated with caveolae. This phenomenon is not observed when a PKC alpha inhibitor is added at the same time. We conclude that the presence of biologically active milli-calpain within myoblast caveolae induces, in a PKC alpha-dependent manner, MARCKS translocation towards the cytosol. Such a localised signalling event may be essential for myoblast fusion and differentiation.

Identification, characterization and phylogenetic signal of an elongation factor‐1 alpha fragment in demosponges (Metazoa, Porifera, Demospongiae)

Erpenbeck, D., Breeuwer, J.A.J. &amp; van Soest, R.W.M. (2005). Identification, characterization and phylogenetic signal of an elongation factor‐1 alpha fragment in demosponges (Metazoa, Porifera, Demospongiae). —Zoologica Scripta, **, *** – ***.Independent molecular markers are needed in order to clarify demosponge systematics. Elongation factor‐1 alpha (EF‐1a) is a frequently used marker in metazoan phylogeny, which has not hitherto been used for Porifera. In this paper, a fragment of the gene encoding EF‐1a is characterized and tested for its suitability in phylogenetic reconstructions of demosponges. We point out the pitfalls in establishing new markers in sponge molecular systematics, particularly with regard to the variety of nonporiferan DNA templates contained within sponges, and show the limited suitability of the fragment for higher demosponge systematics.

Platelet glycoprotein Ib alpha polymorphisms affect the interaction with von Willebrand factor under flow conditions

Interaction of platelet glycoprotein (GP) Ibalpha with von Willebrand factor (VWF) is essential for thrombus formation, particularly under high shear conditions. Previous case-control studies indicated that two GPIb alpha polymorphisms, (145)Thr/Met and/or variable number (1-4) tandem repeats of 13 amino-acid sequences, are associated with arterial thrombosis. The (145)Met-allele and the 3R- or 4R-allele is associated with increased risk. However, there is little clear experimental data to support this association. To elucidate the functional effects of these polymorphisms, we prepared recombinant GPIb alpha fragments and tested them in vitro. The dissociation constants of ristocetin-induced (125)I-labelled VWF binding to two forms of soluble recombinant GPIb alpha [(1)His-(302)Ala, either (145)Thr (145T) or (145)Met (145M)] were not different. Four types of Chinese hamster ovary cells expressing full-length GPIb alpha beta/IX, 145T with one repeat (T1R), 145M with one repeat (M1R), 145T with four repeats (T4R), and 145M with four repeats (M4R), were prepared, and cell interactions with immobilized-VWF were examined under various shear conditions. The cell rolling velocity of M4R under a shear condition of 114/s was significantly slower than that of T1R. Intermediate values were obtained with M1R and T4R. The results suggest that M4R interacts more strongly with VWF under flow conditions.