β‐Blocker in Post‐Myocardial Infarct Survivors with Preserved Left Ventricular Systolic Function

Background: Long‐term β‐blockade therapy is beneficial in post‐myocardial infarct (MI) patients with left ventricular (LV) dysfunction; nevertheless, its benefit in post‐MI patients with preserved LV function remains unclear. The objective of this study is to investigate the effects of long‐term β‐blockade therapy on the clinical outcomes in post‐MI patients with preserved LV function. Hypothesis: The beneficial effects of long‐term β‐blockade therapy in post‐MI patients with impaired LV function may extend to those with preserved LV function. Methods: Of 617 consecutive post‐MI patients referred for cardiac rehabilitation program, 208 patients (age: 62.7 ± 0.8 years; male: 76%) with preserved LV function (ejection fraction ≥ 50%), negative exercise stress test, and on angiotensin‐converting enzyme inhibition were studied. Results: Baseline characteristics were comparable between patients on β‐blocker (n = 154) and not on β‐blocker (n = 54). After a mean follow‐up of 58.5 ± 2.7 months, 14 patients not on β‐blocker (26%) and 14 patients on β‐blocker (9%) died with hazard ratio (HR) of 2.5 (95% confidence interval [CI]: 1.25–6.42, P = 0.01). Likewise, patients not on β‐blocker had a higher incidence of cardiac death (HR: 3.0, 95% CI: 1.07–12.10, P = 0.04), and non‐sudden cardiac death (HR: 10.1, 95% CI: 1.82–89.65, P = 0.01), but not sudden cardiac death compared with patients on β‐blocker (HR: 1.6, 95% CI: 0.34–7.61, P = 0.54). A Cox regression analysis revealed that only advanced age (≥75 years; HR: 2.55, 95% CI: 1.18–5.49, P = 0.02) and the absence of β‐blocker (HR: 2.41, 95% CI: 1.14–5.09, P = 0.02) were independent predictors for mortality. Conclusion: β‐blocker use was associated with a decrease in overall mortality and cardiac death in post‐MI patients with preserved LV function. (PACE 2010; 33:675–680)     

Molecular cloning and characterization of an endogenous digestive β‐glucosidase from the midgut of the fungus‐growing termite Macrotermes barneyi

β‐glucosidase from the midgut of the fungus‐growing termite Macrotermes barneyi was first cloned and characterized to gain a better understanding of cellulolytic systems in fungus‐growing termites. β‐glucosidase activity was proven to present primarily in the midgut of M. barneyi and two β‐glucosidases were partially purified from the midgut. Based on the N‐terminus sequence of one of the β‐glucosidases, a full‐length cDNA fragment of 1708 bp was obtained. This sequence encodes a 493 amino acid protein belonging to glycoside hydrolase family 1. Quantitative real‐time PCR analysis proved that the β‐glucosidase gene was primarily expressed in the midgut. β‐glucosidase was expressed heterologously and biochemically characterized. Results indicate that β‐glucosidase is an endogenous, midgut‐origin termite digestive enzyme. It may have applications in understanding the mechanism of lignocellulose degradation in fungus‐growing termites.     

New definitions of extended‐spectrum β‐lactamase conferring worldwide emerging antibiotic resistance

Although there is no consensus of the precise definition of ESBL, three kinds of ESBL definitions have been proposed. First, the classical definition includes variants derived from TEM‐1, TEM‐2, or SHV‐1; K1 (KOXY) of Klebsiella oxytoca. Second, the broadened definition has stretched the classical definition of ESBL to include: (1) β‐lactamases (CTX‐M‐ESBLs, GES‐ESBLs, and VEB‐ESBLs), with spectra similar to those of TEM and SHV variants (designated as TEM‐ and SHV‐ESBLs, respectively) but derived from other sources; (2) TEM and SHV variants with borderline ESBL activity; e.g., TEM‐12; and (3) various β‐lactamases conferring wider resistance than their parent types but not meeting the definition for group 2be; e.g., OXA‐types (OXA‐ESBLs) and mutant AmpC‐types (AmpC‐ESBLs), with increased activity against oxyimino‐cephalosporins and with resistance to clavulanic acid. Third, the all‐inclusive definition includes: (1) ESBL_A (named for class A ESBLs); (2) ESBL_M (miscellaneous ESBLs), which has been subdivided into ESBL_M‐C (class C; plasmid‐mediated AmpC) and ESBL_M‐D (class D); and (3) ESBL_CARBA (ESBLs with hydrolytic activity against carbapenems), which has been subdivided into ESBL_CARBA‐A (class A carbapenemases), ESBL_CARBA‐B (class B carbapenemases), and ESBL_CARBA‐D (class D carbapenemases). The consensus view about the ESBL definition is that the classical ESBL definition must be expanded to class A non‐TEM‐ and non‐SHV‐ESBLs (CTX‐M‐, GES‐, VEB‐ESBLs, etc.). However, these three definitions evoke rational debate on the question “Which would be included in the category of ESBLs among AmpC‐ESBLs, OXA‐ESBLs, and/or carbapenemases?” Therefore, there is a great need for consensus in the precise definition of ESBL. © 2010 Wiley Periodicals, Inc. Med Res Rev 32:216‐232, 2012     

Prevalence characterization of extended‐spectrum β‐lactamases among Escherichia coli isolates collected in Zhengzhou

Objectives: To determine the prevalence and the diversity of extended‐spectrum β‐lactamases (ESBLs) among Escherichia coli isolates in Zhengzhou, China. Methods: Clinical isolates were collected and investigated from the first affiliated hospital of Zhengzhou University and its associated health‐care facilities in Zhengzhou, China, during the period from January 2006 to June 2008. Antibiograms were performed on Mueller–Hinton agar plates with the disc‐diffusion method and MICs were determined by the agar‐dilution method. Total DNA was extracted with a Qiagen mini kit and screened by PCR. Results: Of 94 nonduplicate ESBL‐positive isolates, TEM‐type was encoded in 74 and 79% of the ESBL isolates. Fifty‐six isolates were SHV type ESBLs. CTX‐M‐1, CTX‐M‐14, CTX‐M‐25, and CTX‐M‐38 types were encoded in 30, 54, 6, and 4, respectively. OXA‐1‐type β‐lactamases were encoded in six and OXA‐20‐type was encoded in two isolates. Conclusions: We describe a complex ESBL epidemiology. The study revealed a high rate of ESBL‐producing E. coli isolates. TEM and CTX‐M enzymes dominated in ESBL‐positive E. coli isolates in Zhengzhou, China. J. Clin. Lab. Anal. 23:404–407, 2009. © 2009 Wiley‐Liss, Inc.     

Integrin Ligands with α/β‐Hybrid Peptide Structure: Design,Bioactivity, and Conformational Aspects

Integrins are cell surface receptors for proteins of the extracellular matrix and plasma‐borne adhesive proteins. Their involvement in diverse pathologies prompted medicinal chemists to develop small‐molecule antagonists, and very often such molecules are peptidomimetics designed on the basis of the short native ligand‐integrin recognition motifs. This review deals with peptidomimetic integrin ligands composed of α‐ and β‐amino acids. The roles exerted by the β‐amino acid components are discussed in terms of biological activity, bioavailability, and selectivity. Special attention is paid to the synthetic accessibility and efficiency of conformationally constrained heterocyclic scaffolds incorporating α/β‐amino acid span.     

An αIIb mutation in patients with Glanzmann thrombasthenia located in the N‐terminus of blade 1 of the β‐propeller (Asn2Asp) disrupts a calcium binding site in blade 6

Summary. Background: Studies of Glanzmann thrombasthenia (GT)‐causing mutations has generated invaluable information on the formation and function of integrin αIIbβ₃. Objective: To characterize the mutation in four siblings of an Israeli Arab family affected by GT, and to analyze the relationships between the mutant protein structure and its function using artificial mutations. Methods and Results: Sequencing disclosed a new A97G transversion in the αIIb gene predicting Asn2Asp substitution at blade 1 of the β‐propeller. Alignment with other integrin α subunits revealed that Asn2 is highly conserved. No surface expression of αIIbβ₃ was found in patients’ platelets and baby hamster kidney (BHK) cells transfected with mutated αIIb and WT β₃. Although the αIIbβ₃ was formed, the mutation impaired its intracellular trafficking. Molecular dynamics simulations and modeling of the αIIbβ₃ crystal indicated that the Asn2Asp mutation disrupts a hydrogen bond between Asn2 and Leu366 of a calcium binding domain in blade 6, thereby impairing calcium binding that is essential for intracellular trafficking of αIIbβ₃. Substitution of Asn2 to uncharged Ala or Gln partially decreased αIIbβ₃ surface expression, while substitution by negatively or positively charged residues completely abolished surface expression. Unlike αIIbβ₃, αVβ₃ harboring the Asn2Asp mutation was surface expressed by transfected BHK cells, which is consistent with the known lower sensitivity of αVβ₃ to calcium chelation compared with αIIbβ₃. Conclusion: The new GT causing mutation highlights the importance of calcium binding domains in the β‐propeller for intracellular trafficking of αIIbβ₃. The mechanism by which the mutation exerts its deleterious effect was elucidated by molecular dynamics.     

Ceftobiprole: a new β‐lactam antibiotic

The increasing threat of antimicrobial resistance in general, and that of methicillin‐resistant Staphylococcus aureus (MRSA) in particular, is raising significant medical, economical and public health challenges worldwide, both within hospitals and throughout the community. These considerations, along with the extensive time and costs associated with the development and approval of new therapeutic agents, represent some of the major reasons why understanding the advantages and limitations of new antibiotics, ensuring their judicious use and maximising their active shelf life should become global priorities. On March 18, 2008, the Food and Drug Administration issued an approvable letter for ceftobiprole, a broad‐spectrum β‐lactam antibiotic active against MRSA and other clinically relevant Gram‐positive and Gram‐negative pathogens. Ceftobiprole is currently available only for parenteral administration, and besides its remarkable antimicrobial spectrum, this antibiotic possesses additional desirable characteristics, such as low propensity to select for resistance, efficacy in animal models of disease and good safety profile. Furthermore, in recently completed clinical trials, ceftobiprole demonstrated non‐inferiority to comparator compounds such as vancomycin, and emerged as a promising clinical option of monotherapy for the treatment of complicated skin and skin structure infections and community‐acquired pneumonia. Here, we discuss some of the most important clinically relevant findings on ceftobiprole obtained from in vitro studies, animal models of disease and recently completed phase III clinical trials.     

Antispasmodic effects of essential oil of Pterodon polygalaeflorus and its main constituent β‐caryophyllene on rat isolated ileum

This study investigates the effects of essential oil of Pterodon polygalaeflorus (EOPP) and β‐caryophyllene (β‐CAR). EOPP and β‐CAR relaxed the basal tone of ileum smooth muscle in a concentration‐dependent manner (IC₅₀s = 394.35 ± 62.12 and 68.65 ± 9.51 μg/mL respectively), an effect that was unaltered by hexamethonium, L ‐nitroarginine methyl ester or indomethacin. Both EOPP and β‐CAR evoked a concentration‐dependent relaxation of ileum pre‐contracted with KCl with an IC₅₀ value of 107.78 ± 10.47 and 17.35 ± 0.75 μg/mL, respectively. EOPP and β‐CAR inhibited the contractions induced by acetylcholine (ACh) and by KCl. In ileal preparations, the CaCl₂‐induced contractions were reduced by EOPP (300 μg/mL) and β‐CAR (100 μg/mL). Furthermore, CaCl₂‐induced contractions were also reduced by EOPP (300 μg/mL) and β‐CAR (100 μg/mL) in ileal preparations pretreated with ACh under Ca²⁺‐free condition and in the presence of verapamil. EOPP (100 and 300 μg/mL) and β‐CAR (30 and 100 μg/mL) reduced the ACh‐induced contractions of isolated rat ileum under Ca²⁺‐free conditions. In the presence of high KCl and Ca²⁺‐free conditions, EOPP (300 μg/mL) and β‐CAR (100 μg/mL) reduced the contractions induced by barium. A similar effect was also observed with verapamil. It is concluded that (i) β‐CAR is an important constituent involved in the myorelaxant and antispasmodic effects induced by EOPP; (ii) the inhibitory effect on intestinal contractility is myogenic and seems mainly mediated through an intracellular mechanism. However, the ability of EOPP and β‐CAR to decrease Ca²⁺ influx through cytoplasmic membrane could not be discounted.     

β2‐Glycoprotein I: evolution,structure and function

Summary. β₂‐Glycoprotein I (β₂‐GPI) is a protein that circulates in blood at high concentrations. The function of β₂‐GPI has long been an enigma. More than 20 years ago, it was discovered that β₂‐GPI is the major antigen for the circulating antibodies in the antiphospholipid syndrome. However, this knowledge has not advanced our understanding of the physiologic role of the protein. In recent years, new insights have suggested an important function of this protein in innate immunity. β₂‐GPI was found to scavenge lipopolysaccharide and was able to clear unwanted anionic cellular remnants such as microparticles from the circulation. The function of β₂‐GPI seems to depend on the structural conformation of the protein, and it has been established that β₂‐GPI can exist in at least two conformations. In this review, we will highlight and summarize the current knowledge on this protein.     

The calcification potential of human MSCs can be enhanced by interleukin‐1β in osteogenic medium

Inflammation is one of the key regulators of the repair process in bone tissues. Current data about the effect of interleukin‐1β (IL‐1β) on MSCs and osteoblasts are conflicting. We investigated the long‐term effect of IL‐1β on direct osteogenic differentiation of hMSCs in vitro. IL‐1β‐stimulated cells showed enhanced proliferation and entered maturation prior to non‐stimulated ones, as monitored by ALP activity. The process of calcification was accelerated during long‐term stimulation of hMSCs with IL‐1β. Since donor variability is a well‐known issue, we suggest a new method to illustrate global changes of a random chosen donor population through collative analysis. We further demonstrate an absorbance assay to evaluate the degree of calcification during in vitro culture of monolayer expanded hMSCs. Our findings support the importance of IL‐1β in osteogenic differentiation of hMSCs in an in vitro monolayer culture model. A new online absorbance assay is a useful method to evaluate the osteogenic differentiation of hMSCs at early stages. These findings will be helpful in optimizing predifferentiation of hMSCs in vitro for bone tissue engineering. Copyright © 2014 John Wiley & Sons, Ltd.     

Vicinal thiols are required for activation of the αIIbβ3 platelet integrin

Summary. Background: Closely spaced thiols in proteins that interconvert between the dithiol form and disulfide bonds are called vicinal thiols. These thiols provide a mechanism to regulate protein function. We previously found that thiols in both αIIb and β3 of the αIIbβ3 fibrinogen receptor were required for platelet aggregation. Methods and Results: Using p‐chloromercuribenzene sulfonate (pCMBS) we provide evidence that surface thiols in αIIbβ3 are exposed during platelet activation. Phenylarsine oxide (PAO), a reagent that binds vicinal thiols, inhibits platelet aggregation and labeling of sulfhydryls in both αIIb and β3. For the aggregation and labeling studies, binding of PAO to vicinal thiols was confirmed by reversal of PAO binding with the dithiol reagent 2,3‐Dimercapto‐1‐propanesulfonic acid (DMPS). In contrast, the monothiol β‐mercaptoethanol did not reverse the effects of PAO. Additionally, PAO did not inhibit sulfhydryl labeling of the monothiol protein albumin, confirming the specificity of PAO for vicinal thiols in αIIbβ3. As vicinal thiols represent redox sensitive sites that can be regulated by reducing equivalents from the extracellular or cytoplasmic environment, they are likely to be important in regulating activation of αIIbβ3. Additionally, when the labeled integrin was passed though a lectin column containing wheat germ agglutinin and lentil lectin a substantial amount of non‐labeled αIIbβ3 eluted separately from the labeled receptor. This suggests that two populations of integrin exist on platelets that can be distinguished by thiol labeling. Conclusion: A vicinal thiol‐containing population of αIIbβ3 provides redox sensitive sites for regulation of αIIbβ3.     

Induction of anti‐β2‐glycoprotein I autoantibodies in mice by protein H of Streptococcus pyogenes

Summary. Background: The antiphospholipid syndrome (APS) is characterized by the persistent presence of anti‐β₂‐glycoprotein I (β₂‐GPI) autoantibodies. β₂‐GPI can exist in two conformations. In plasma it is a circular protein, whereas it adopts a fish‐hook conformation after binding to phospholipids. Only the latter conformation is recognized by patient antibodies. β₂‐GPI has been shown to interact with Streptococcus pyogenes. Objective: To evaluate the potential of S. pyogenes‐derived proteins to induce anti‐β₂‐GPI autoantibodies. Methods and results: Four S. pyogenes surface proteins (M1 protein, protein H, streptococcal collagen‐like protein A [SclA], and streptococcal collagen‐like protein B [SclB]) were found to interact with β₂‐GPI. Only binding to protein H induces a conformational change in β₂‐GPI, thereby exposing a cryptic epitope for APS‐related autoantibodies. Mice were injected with the four proteins. Only mice injected with protein H developed antibodies against the patient antibody‐related epitope in domain I of β₂‐GPI. Patients with pharyngotonsillitis caused by S. pyogenes who developed anti‐protein H antibodies also generated anti‐β₂‐GPI antibodies. Conclusions: Our study has demonstrated that a bacterial protein can induce a conformational change in β₂‐GPI, resulting in the formation of antiβ₂‐GPI autoantibodies. This constitutes a novel mechanism for the formation of anti‐β₂‐GPI autoantibodies.     

Effects of variation in the human α2A‐ and α2C‐adrenoceptor genes on cognitive tasks and pain perception

Background: The mechanisms underlying interindividual variability in pain perception and cognitive responses are undefined but highly heritable. α_2C‐ and α_2A‐adrenergic receptors regulate noradrenergic activity and are important mediators of pain perception and analgesia. We hypothesized that common genetic variants in these genes, particularly the ADRA2C 322–325 deletion variant, affect pain perception or cognitive responses. Methods: We studied 73 healthy subjects (37 Caucasians and 36 African–Americans) aged 25.4 ± 4.6 years. Pain response to a cold pressor test was measured using a 10 cm visual analog scale and again on the next day, after three infusions of the selective α₂‐agonist dexmedetomidine. Standardized cognitive tests were administered at baseline and after each infusion. The contribution of ADRA2C deletion genotype, dexmedetomidine concentration, and other covariates to pain perception and cognitive responses was determined using multiple linear regression models. Secondary analysis examined the effects of ADRA2A and other ADRA2C variants on pain perception. Results: ADRA2C Del homozygotes had higher pain scores in response to cold at baseline (6.3 ± 1.8 cm) and after dexmedetomidine (5.6 ± 2.2 cm) than insertion allele carriers (4.6 ± 2.1 cm [baseline] and 3.8 ± 1.9 cm [after dexmedetomidine]; adjusted P‐values = 0.019 and 0.004, respectively). Cognitive responses were unrelated to ADRA2C Ins/Del genotype. None of the other ADRA2A and ADRA2C variants was significantly related to cold pain sensitivity before dexmedetomidine; after dexmedetomidine, ADRA2A rs1800038 was marginally associated (P = 0.03). Conclusion: The common ADRA2C del322–325 variant affected pain perception before and after dexmedetomidine but did not affect other cognitive responses, suggesting that it contributes to interindividual variability in pain perception.     

In vivo and ex vivo 19‐fluorine magnetic resonance imaging and spectroscopy of beta‐cells and pancreatic islets using GLUT‐2 specific contrast agents

The assessment of the β‐cell mass in experimental models of diabetes and ultimately in patients is a hallmark to understand the relationship between reduced β‐cell mass/function and the onset of diabetes. It has been shown before that the GLUT‐2 transporter is highly expressed in both β‐cells and hepatocytes and that D‐mannoheptulose (DMH) has high uptake specificity for the GLUT‐2 transporter. As 19‐fluorine MRI has emerged as a new alternative method for MRI cell tracking because it provides potential non‐invasive localization and quantification of labeled cells, the purpose of this project is to validate β‐cell and pancreatic islet imaging by using fluorinated, GLUT‐2 targeting mannoheptulose derivatives (¹⁹FMH) both in vivo and ex vivo. In this study, we confirmed that, similar to DMH, ¹⁹FMHs inhibit insulin secretion and increase the blood glucose level in mice temporarily (approximately two hours). We were able to assess the distribution of ¹⁹FMHs in vivo with a temporal resolution of about 20 minutes, which showed a quick removal of ¹⁹FMH from the circulation (within two hours). Ex vivo MR spectroscopy confirmed a preferential uptake of ¹⁹FMH in tissue with high expression of the GLUT‐2 transporter, such as liver, endocrine pancreas and kidney. No indication of further metabolism was found. In summary, ¹⁹FMHs are potentially suitable for visualizing and tracking of GLUT‐2 expressed cells. However, current bottlenecks of this technique related to the quick clearance of the compound and relative low sensitivity of ¹⁹F MRI need to be overcome. Copyright © 2016 John Wiley & Sons, Ltd.     

Collagen‐mimetic peptides mediate flow‐dependent thrombus formation by high‐ or low‐affinity binding of integrin α2β1 and glycoprotein VI

Summary. Background: Collagen acts as a potent surface for platelet adhesion and thrombus formation under conditions of blood flow. Studies using collagen‐derived triple‐helical peptides have identified the GXX’GER motif as an adhesive ligand for platelet integrin α2β1, and (GPO)_n as a binding sequence for the signaling collagen receptor, glycoprotein VI (GPVI). Objective: The potency was investigated of triple‐helical peptides, consisting of GXX’GER sequences within (GPO)_n or (GPP)_n motifs, to support flow‐dependent thrombus formation. Results: At a high‐shear rate, immobilized peptides containing both the high‐affinity α2β1‐binding motif GFOGER and the (GPO)_n motif supported platelet aggregation and procoagulant activity, even in the absence of von Willebrand factor (VWF). With peptides containing only one of these motifs, co‐immobilized VWF was needed for thrombus formation. The (GPO)_n but not the (GPP)_n sequence induced GPVI‐dependent platelet aggregation and procoagulant activity. Peptides with intermediate affinity (GLSGER, GMOGER) or low‐affinity (GASGER, GAOGER) α2β1‐binding motifs formed procoagulant thrombi only if both (GPO)_n and VWF were present. At a low‐shear rate, immobilized peptides with high‐ or low‐affinity α2β1‐binding motifs mediated formation of thrombi with procoagulant platelets only in combination with (GPO)_n. Conclusions: Triple‐helical peptides with specific receptor‐binding motifs mimic the properties of native collagen I in thrombus formation by binding to both platelet collagen receptors. At a high‐shear rate, either GPIb or high‐affinity (but not low‐affinity) GXX’GER mediates GPVI‐dependent formation of procoagulant thrombi. By extension, high‐affinity binding for α2β1 can control the overall platelet‐adhesive activity of native collagens.     

The activin‐βA/BMP‐2 chimera AB204 is a strong stimulator of adipogenesis

Several of the bone morphogenetic proteins (BMPs) have been reported to induce white as well as brown adipogenesis. Here, we characterized the adipogenic potential of AB204, a recombinant chimeric protein of activin‐βA and BMP‐2, in in vitro, ex vivo and in vivo settings. BMP‐2 is generally known to promote adipogenesis. When compared with BMP‐2, which previously showed varying degrees of adipogenesis, AB204 displayed superior in vitro adipogenic differentiation of mouse 3 T3‐L1 pre‐adipocytes and human adipose‐derived stem cells (hASCs). Surprisingly, implantation of hASCs, preconditioned with AB204 for as short a time as 48 h, into the subcutaneous space of athymic nude mice effectively produced fat pads, but not with BMP‐2. When BMP‐2 and AB204 were injected intraperitoneally, AB204 promoted dramatic systemic adipogenesis of C57BL/6 mice on a high‐fat diet very effectively. The results implicate the novel clinical potential of AB204, including induction of fat tissue ex vivo or in vivo for tissue re‐engineering and regenerative medicinal purposes, more than any known natural protein ligand. Copyright © 2015 John Wiley & Sons, Ltd.     

Optimization of culture conditions for osteogenically‐induced mesenchymal stem cells in β‐tricalcium phosphate ceramics with large interconnected channels

The aim of this study was to optimize culture conditions for human mesenchymal stem cells (hMSCs) in β‐tricalcium phosphate ceramics with large interconnected channels. Fully interconnected macrochannels comprising pore diameters of 750 µm and 1400 µm were inserted into microporous β‐tricalcium phosphate (β‐TCP) scaffolds by milling. Human bone marrow‐derived MSCs were seeded into the scaffolds and cultivated for up to 3 weeks in both static and perfusion culture in the presence of osteogenic supplements (dexamethasone, β‐glycerophosphate, ascorbate). It was confirmed by scanning electron microscopic investigations and histological staining that the perfusion culture resulted in uniform distribution of cells inside the whole channel network, whereas the statically cultivated cells were primarily found at the surface of the ceramic samples. It was also determined that perfusion with standard medium containing 10% fetal calf serum (FCS) led to a strong increase (seven‐fold) of cell numbers compared with static cultivation observed after 3 weeks. Perfusion with low‐serum medium (2% FCS) resulted in moderate proliferation rates which were comparable to those achieved in static culture, although the specific alkaline phosphatase (ALP) activity increased by a factor of more than 3 compared to static cultivation. Gene expression analysis of the ALP gene also revealed higher levels of ALP mRNA in low‐serum perfused samples compared to statically cultivated constructs. In contrast, gene expression of the late osteogenic marker bone sialoprotein II (BSPII) was decreased for perfused samples compared to statically cultivated samples. Copyright © 2010 John Wiley & Sons, Ltd.     

A role for adhesion and degranulation‐promoting adapter protein in collagen‐induced platelet activation mediated via integrin α2β1

Summary. Background: Collagen‐induced platelet activation is a key step in the development of arterial thrombosis via its interaction with the receptors glycoprotein (GP)VI and integrin α₂β₁. Adhesion and degranulation‐promoting adapter protein (ADAP) regulates α_IIbβ₃ in platelets and α_Lβ₂ in T cells, and is phosphorylated in GPVI‐deficient platelets activated by collagen. Objectives: To determine whether ADAP plays a role in collagen‐induced platelet activation and in the regulation and function of α₂β₁. Methods: Using ADAP^?/? mice and synthetic collagen peptides, we investigated the role of ADAP in platelet aggregation, adhesion, spreading, thromboxane synthesis, and tyrosine phosphorylation. Results and Conclusions: Platelet aggregation and phosphorylation of phospholipase Cγ2 induced by collagen were attenuated in ADAP^?/? platelets. However, aggregation and signaling induced by collagen‐related peptide (CRP), a GPVI‐selective agonist, were largely unaffected. Platelet adhesion to CRP was also unaffected by ADAP deficiency. Adhesion to the α₂β₁‐selective ligand GFOGER and to a peptide (III‐04), which supports adhesion that is dependent on both GPVI and α₂β₁, was reduced in ADAP^?/? platelets. An impedance‐based label‐free detection technique, which measures adhesion and spreading of platelets, indicated that, in the absence of ADAP, spreading on GFOGER was also reduced. This was confirmed with non‐fluorescent differential‐interference contrast microscopy, which revealed reduced filpodia formation in ADAP^?/? platelets adherent to GFOGER. This indicates that ADAP plays a role in mediating platelet activation via the collagen‐binding integrin α₂β₁. In addition, we found that ADAP^?/? mice, which are mildly thrombocytopenic, have enlarged spleens as compared with wild‐type animals. This may reflect increased removal of platelets from the circulation.