Subject-specific,whole-body models of the stooped posture with a personal weight transfer device

A prior laboratory study found that when wearing a weight transfer device in the stooped posture, trunk flexions were reduced, and subjects who did not experience flexion–relaxation of the erector spinae had reduced back muscle activity. Whole-body musculoskeletal models, which included individual passive torso stiffness and anthropometry, were implemented to predict loads in the passive tissues of the back and the leg joints. Results predicted that when wearing the device in the stooped posture, compression and shear forces at the L5–S1 level were reduced by 13% and 12% respectively. Internal loads in the leg joints were reduced between 10% and 31%. Much of the reduction in joint loads may be a result of the device’s ability to limit torso flexion during stoop, rather than a transferring of load. While these results show possible benefit in the short-term, further study is needed on the long-term effects to determine if the device is an effective intervention for those who use the stooped posture routinely.     

Prevalence of MTHFR C677T Single Nucleotide Polymorphism in Genetically Isolated Populations in Jordan

Methylenetetrahydrofolate reductase (MTHFR) C677T single nucleotide polymorphism is a major inherited risk factor of venous thromboembolism. We sought to determine its prevalence in genetically isolated populations of Chechens and Circassians in Jordan. The MTHFR C677T mutation was analyzed from blood samples taken from 120 random unrelated Chechens and 72 Circassians. The prevalence of the MTHFR mutation in the Chechen population was 27.5% (allele frequency 15%); the prevalence among the Circassians was 50% (allele frequency 29.2%). The prevalence in the Chechen population is similar to that in Jordan and other world populations, but it is higher in the Circassian population. This study will contribute to understanding the interaction between genetic and environmental risk factors underlying thrombosis and will be useful in deciding which genetic variants should be tested in a clinical genetic testing service.     

Maurotoxin versus Pi1/HsTx1 scorpion toxins. Toward new insights in the understanding of their distinct disulfide bridge patterns

Maurotoxin (MTX) is a scorpion toxin acting on several K(+) channel subtypes. It is a 34-residue peptide cross-linked by four disulfide bridges that are in an "uncommon" arrangement of the type C1-C5, C2-C6, C3-C4, and C7-C8 (versus C1-C5, C2-C6, C3-C7, and C4-C8 for Pi1 or HsTx1, two MTX-related scorpion toxins). We report here that a single mutation in MTX, in either position 15 or 33, resulted in a shift from the MTX toward the Pi1/HsTx1 disulfide bridge pattern. This shift is accompanied by structural and pharmacological changes of the peptide without altering the general alpha/beta scaffold of scorpion toxins.     

Analysis of the interacting surface of maurotoxin with the voltage‐gated Shaker B K+ channel

Maurotoxin (MTX) is a 34‐residue toxin that was isolated initially from the venom of the scorpion Scorpio maurus palmatus. Unlike the other toxins of the α‐KTx6 family (Pi1, Pi4, Pi7, and HsTx1), MTX exhibits a unique disulfide bridge organization of the type C₁? C₅, C₂? C₆, C₃? C₄, and C₇? C₈ (instead of the conventional C₁? C₅, C₂? C₆, C₃? C₇, and C₄? C₈, herein referred to as Pi1‐like) that does not prevent its folding along the classic α/β scaffold of scorpion toxins. MTX_Pi1 is an MTX variant with a conventional pattern of disulfide bridging without any primary structure alteration of the toxin. Here, using MTX and/or MTX_Pi1 as models, we investigated how the type of folding influences toxin recognition of the Shaker B potassium channel. Amino acid residues of MTX that were studied for Shaker B recognition were selected on the basis of their homologous position in charybdotoxin, a three disulfide‐bridged scorpion toxin also active on this channel type. These residues favored either an MTX‐ or MTX_Pi1‐like folding. Our data indicate clearly that Lys²³ and Tyr³² (two out of ten amino acid residues studied) are the most important residues for Shaker B channel blockage by MTX. For activity on SKCa channels, the same amino acid residues also affect, directly or indirectly, the recognition of SK channels. The molecular modeling technique and computed docking indicate the existence of a correlation between the half cystine pairings of the mutated analogs and their activity on the Shaker B K⁺ channel. Overall, mutations in MTX could, or could not, change the reorganization of disulfide bridges of this molecule without affecting its α/β scaffold. However, changing of the peptide backbone (cross linking disulfide bridges from MTX‐like type vs MTX_Pi1‐like type) appears to have less impact on the molecule activity than mutation of certain key amino acids such as Lys²³ and Tyr³² in this toxin. Copyright © 2011 European Peptide Society and John Wiley & Sons, Ltd.     

Use of Taguchi's methods as a basis to optimize hybridoma cell line growth and antibody production in a spinner flask

Taguchi’s methods were used for the design of an experimental strategy aimed at optimizing cell density and monoclonal antibody (mAb) production from a spinner flask hybridoma culture. 23G11 is an antibody to the human leukocyte adhesion molecule, CR3 or β 2 integrin (CD11b/CD18). It recognizes specifically the A-domain of the α subunit CD11b. Anti β 2 integrin monoclonal antibodies hold a great potential for preventing inflammation mediated tissue injuries. An L8 orthogonal experimental design was used to investigate four different culture components: stirring speed, nature of serum, concentration of serum and nature of media (RPMI 1640 or RPMI 1640 supplemented with glucose and glutamine). The experiments were conducted using two levels for each factor studied and a direct ELISA test was used to estimate the level of antibody production. Statistical analysis of the collected data pointed to the stirring speed and serum concentration, and the interaction between these parameters, as the components that affected cell growth. Antibody production was affected by these factors and by the nature of medium but also by the following two interactions: stirring speed/nature of serum and stirring speed/concentration of serum. This study emphasizes the value of using Taguchi’s methods as a basis for optimization of mAb production from a hybridoma culture, in cost effective and significantly less labor intensive ways. This revised version was published online in August 2006 with corrections to the Cover Date.     

Effects of PKCalpha activation on Ca2+ pump and KCa channel in deoxygenated sickle cells

We have previously shown that a pretreatment with phorbol12-myristate 13-acetate (PMA), an activator of protein kinase C (PKC),reduced deoxygenation-induced K⁺loss and Ca²⁺ uptake and preventedcell dehydration in sickle anemia red blood cells (SS cells) (H. Fathallah, E. Coezy, R.-S. De Neef, M.-D. Hardy-Dessources, and F. Giraud. Blood 86: 1999-2007,1995). The present study explores the detailed mechanism of thisPMA-induced inhibition. The main findings are, first, the detection ofPKC and PKC in normal red blood cells and the demonstration that both isoforms are expressed at higher levels in SS cells. The -isoform only is translocated to the membrane and activated by PMAand by elevation of cytosolicCa²⁺. Second, PMA is demonstratedto activate Ca²⁺ efflux indeoxygenated SS cells by a direct stimulation of the Ca²⁺ pump. PMA, moreover, inhibitsdeoxygenation-induced, charybdotoxin-sensitive K⁺ efflux in SS cells. Thisinhibition is partly indirect and explained by the reduceddeoxygenation-induced rise in cytosolicCa²⁺ resulting fromCa²⁺ pump stimulation. However, asignificant inhibition of theCa²⁺-activatedK⁺ channels(K_Ca channels) by PMA can also bedemonstrated when the channels are activated byCa²⁺ plus ionophore, underconditions in which the Ca²⁺ pumpis operating near its maximal extrusion rate, but swamped byCa²⁺ plus ionophore. The data thussuggest a PKC-mediated phosphorylation both of theCa²⁺ pump and of theK_Ca channel or an auxiliaryprotein.

     

Cloning of the rat CR3 alphaM (CD11b) subunit, expression and binding assay of recombinant isolated CD11b VA (A-domain) and ICAM-1 Ig modules

The leukocyte beta2 integrin CR3 (CD11/CD18), is a surface heterodimeric glycoprotein that functions as a divalent cation-dependent adhesive complex. It mediates several important cell-substrate and cell-cell adhesive interactions among which the interaction with vascular endothelial cells that lead to leukocyte transmigration. We have isolated cDNA clones-coding for the rat complement receptor type 3 (CR3) alphaM subunit (CD11b) from a cDNA library. The cDNA sequence showed respectively 89.4% and 74.6% homology with its mouse and human counterpart. We have expressed the sequence coding for the VA module or Von Willebrand type domain (A-domain) and produced it in E. coli as a soluble recombinant fusion protein with GST. Simultaneously, we have cloned DNA fragments specific to the rat ICAM-1 domain 1 and domain 3 and expressed each clone in E. coli as recombinant soluble (rs) fusion proteins with GST. Recombinant CD11b A-domain was released from the fusion protein by thrombin cut. Purified ICAM-1 fusion peptides and CD11b A-domain were used to develop a direct binding assay that showed a specific binding between the rat ICAM-1 Ig like domain 3 and CD11b A-domain. These data demonstrate that the IgSF modules can be produced as a soluble recombinant fusion protein and used to study direct binding to the VA module displayed by members of the integrin superfamily.     

Deoxygenation of sickle red blood cells stimulates KCl cotransport without affecting Na+/H+ exchange

KCl cotransport activated by swelling of sickle red blood cells(SS RBC) is inhibited by deoxygenation. Yet recent studies found aCl-dependent increase insickle reticulocyte density with cyclic deoxygenation. This studysought to demonstrate cotransporter stimulation by deoxygenation of SSRBC in isotonic media with normal pH. Low-density SS RBC exhibited aCl-dependent component ofthe deoxygenation-induced net K⁺efflux, which was blocked by two inhibitors of KCl cotransport, [(dihydroindenyl)oxy]alkanoic acid and okadaic acid.Cl-dependentK⁺ efflux stimulated bydeoxygenation was enhanced 2.5-fold by clamping of cellularMg²⁺ at the level in oxygenatedcells using ionophore A-23187. Incubating cells in high externalK⁺ orRb⁺ minimized inhibition of KClcotransport by internal Mg²⁺, andunder these conditions deoxygenation markedly stimulated KClcotransport in the absence of ionophore. Activation of KCl cotransportby deoxygenation of SS RBC in isotonic media at normal pH is consistentwith the generalized dephosphorylation of membrane proteins induced bydeoxygenation and activation of the cotransporter by adephosphorylation mechanism.Na⁺/H⁺exchange activity, known to be modulated by cytosolicCa²⁺ elevation and cell shrinkage,remained silent under deoxygenation conditions.

     

A unique mutation underlying carbonic anhydrase II deficiency syndrome in patients of Arab descent

We have investigated, in the genomic DNA of ten Tunisian patients, the presence of a splice junction mutation at the 5 end of intron 2 in the carbonic anhydrase II gene (CAII) previously described in six CAII-deficient patients presumed to be of Arab origin. All our patients were homozygous for this mutation and were mentally retarded, a characteristic feature of the phenotype of patients with an Arabic background. This mutation is found exclusively in patients with an Arabic background and thus may be confined to this ethnic group.