Site-directed mutagenesis of the acute-phase human serum amyloid A (SAA1 alpha) protein was used to evaluate the importance of the N-terminal amino acid residues, namely RSFFSFLGEAF The full-length cDNA clone of SAA1 alpha (pA1.mod.) was used to create two mutations, namely Gly-8 to Asp-8 and an 11 amino acid truncation between Arg-1 and Phe-11 respectively. Wild-type and mutant cDNAs were expressed in Chinese hamster ovary (CHO) cells under the control of the human cytomegalovirus promoter, which resulted in the secretion of the processed proteins into the culture media. Wild-type recombinant human SAA (rSAA) protein was shown to have pI values of 6.0 and 6.4, similar to the human SAA isoform SAA1 alpha and SAA1 alpha desArg found in acute-phase plasma. N-terminal sequencing of 56 residues confirmed its identity with human SAA1 alpha. The total yield of wild-type rSAA measured by ELISA was between 3.5 and 30 mg/l. The two mutations resulted in reduced expression levels of the mutant SAA proteins (3-10 mg/l). Further measurements of rSAA concentration in lipid fractions of culture medium collected at a density of 1.21 g/ml (high-density liporotein; HDL) and 1.063-1.18 g/ml (very-low-density lipoprotein/low-density lipoprotein; VLDL/LDL) showed that 76% of the wild-type protein was found in the HDL fraction and the remaining 24% in the infranatant non-lipid fraction. In contrast the relative concentration of mutant rSAA in HDL and infranatant fractions was reversed. This is consistent with the previously proposed involvement of the 11 amino acid peptide in anchoring. SAA protein on to HDL3 [Turnell, Sarra, Glover, Baum, Caspi, Baltz and Pepys (1986) Mol. Biol. Med. 3, 387-407]. Wild-type rSAA protein was shown to from amyloid fibrils in vitro under acidic conditions as shown by electron microscopy, and stained positive with Congo Red and exhibited apple-green birefringence when viewed under polarized light. Under the same conditions mutSAA(G8D) and mutSAA delta 1-11 did not form amyloid fibrils. In conclusion, replacement of Gly-8 by Asp-8 or deletion of the first 11 amino acid residues at the N-terminus of rSAA diminishes its capacity to bind to HDL and decreases amyloid fibril formation.
The gamma-carboxyglutamic acid (Gla) domains of the vitamin K-dependent blood coagulation proteins contain 10 highly conserved Gla residues within the first 33 residues, but factor IX is unique in possessing 2 additional Gla residues at positions 36 and 40. To determine their importance, factor IX species lacking these Gla residues were isolated from heterologously expressed human factor IX. Using ion-exchange chromatography, peptide mapping, mass spectrometry, and N-terminal sequencing, we have purified and identified two partially carboxylated recombinant factor IX species; factor IX/gamma 40E is uncarboxylated at residue 40 and factor IX/gamma 36,40E is uncarboxylated at both residues 36 and 40. These species were compared with the fully gamma-carboxylated recombinant factor IX, unfractionated recombinant factor IX, and plasma-derived factor IX. As monitored by anti-factor IX:Ca (II)-specific antibodies and by the quenching of intrinsic fluorescence, all these factor IX species underwent the Ca(II)-induced conformational transition required for phospholipid membrane binding and bound equivalently to phospholipid vesicles composed of phosphatidylserine, phosphatidylcholine, and phosphatidylethanolamine. Endothelial cell binding was also similar in all species, with half-maximal inhibition of the binding of 125I-labeled plasma-derived factor IX at concentrations of 2-6 nM. Functionally, factor IX/gamma 36,40E and factor IX/gamma 40E were similar to fully gamma-carboxylated recombinant factor IX and plasma-derived factor IX in their coagulant activity and in their ability to participate in the activation of factor X in the tenase complex both with synthetic phospholipid vesicles and activated platelets. However, Gla 36 and Gla 40 represent part of the epitope targeted by anti-factor IX:Mg(II)-specific antibodies because these antibodies bound factor IX preferentially to factor IX/gamma 36,40E and factor IX/gamma 40E. These results demonstrate that the gamma-carboxylation of glutamic acid residues 36 and 40 in human factor IX is not required for any function of factor IX examined.
BackgroundFacebook is a social networking service launched in February 2004, owned and operated by Facebook, Inc. As of June 2012, Facebook reports more than 1 billion active users. Objective of study was to evaluate the effect of Facebook on the social life, health and behavior of medical students.MethodologyIt was a cross sectional, observational and questionnaire based study conducted in Dow University OF Health Sciences during the period of January 2012 to November 2012. We attempted to interview all the participants who could be approached during the period of the study. Participants were MBBS students, while all students of other courses and programs were taken as exclusion criteria. Approximately 1050 questionnaires were distributed to participants. Fifty questionnaires were rejected due to incomplete answers, yielding 1000 usable responses for an approximate 95% response rate. Informed verbal consent was taken from each participant. Study was ethically approved by Institutional Review Board of Dow University of Health Sciences. All the data was entered and analyzed through SPSS 19.ResultOut of total 1000 participants, males were 400 (40%) and females were 600 (60%). Participants were in the age group of 18–25 years with a mean age of 20.08 years. Most of the participants were using Facebook daily (N = 640, 64%) for around 3–4 hours (N = 401, 40.1%). Majority of them (N = 359, 35.9%) believed that they were equally active on Facebook and in real life while few believed their social life became worse after start using Facebook (N = 372, 37.2%). Most of the participants admitted that they were considered as shy in real world (N = 390, 39.0%) while in the world of Facebook they were considered as fun loving by their friends (N = 603, 60.3%). A large number of participants (N = 715, 75%) complained of mood swings.ConclusionYoungsters are willing to compromise their health, social life, studies for the sake of fun and entertainment or whatever satisfaction they get after using Facebook. What we observed in our study was that although majority of our subjects showed multiple signs of Facebook addiction, they don’t realize it and if even they realize it they don’t want to quit Facebook and even if they want to quit, they can’t. Our observance concluded that majority of the users are highly addicted.
Coagulation factor VIII (FVIII) is an important glycoprotein co-factor involved in haemostasis, functioning to accelerate activation of factor X by activated factor IX. Insertion of expression vectors containing the full-length cDNA sequence of human FVIII into mammalian cell lines results in the production of recombinant factor VIII (rFVIII), typically referred to as 'full-length' rFVIII (FLrFVIII). Both FLrFVIII and plasma-derived FVIII exist primarily as heterodimeric proteins, consisting of a heterogenous light and heavy chain. The objectives of this study were to compare the structural heterogeneity of high-purity FVIII preparations and further define the term 'full length' as it refers to rFVIII protein structure. Five commercially available FVIII concentrates were characterized based on SDS-PAGE, N-terminal sequencing, and peptide and domain mapping coupled to mass spectrometry. The major heavy chain species identified in FLrFVIII included various B-domain-truncated forms of FVIII, with the predominant species terminating at Arg(1313). This study demonstrates that the use of full-sequence FVIII cDNA for the production of rFVIII does not result in a homogeneous FLrFVIII protein product. Rather, commercially available FLrFVIII represents a heterogenous mixture of various B-domain-truncated forms of the molecule, with no evidence of a contiguous, intact B-domain.
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