Effects of second-codon mutations on expression of the insulin-like growth factor-II-encoding gene in Escherichia coli

Expression plasmids encoding random sequence mutant proteins of insulin-like growth factor II (IGFII) were constructed by cassette mutagenesis, to improve the efficiency of IGFII synthesis in Escherichia coli. A pool of oligodeoxyribonucleotide linkers containing random trinucleotide sequences were used to introduce second-codon substitutions into the gene encoding Met-Xaa-Trp-IGFII in expression vectors. E. coli RV308 cells transformed with these vectors synthesized IGFII at levels varying from 0-22% of total cell protein. This variable synthesis is a function of the random second-codon sequence and its corresponding amino acid, Xaa. Our data showed that mRNA stability, protein stability and translational efficiency all contributed to variable expression levels of Met-Xaa-Trp-IGFII in E. coli. Furthermore, an efficiently synthesized IGFII mutant protein, Met-His-Trp-IGFII, was converted to natural sequence IGFII by a simple oxidative cleavage reaction.     

Review: Diagnosis and treatment of dementia: 1. Risk assessment and primary prevention of Alzheimer disease

Background

In addition to nonmodifiable genetic risk factors, potentially modifiable factors such as hypertension, hyperlipidemia and environmental exposures have been identified as risk factors for Alzheimer disease. In this article, we provide physicians with practical guidance on risk assessment and primary prevention of Alzheimer disease based on recommendations from the Third Canadian Consensus Conference on the Diagnosis and Treatment of Dementia, held in March 2006.

Methods

We developed evidence-based guidelines using systematic literature searches, with specific criteria for study selection and quality assessment, and a clear and transparent decision-making process. We selected studies published from January 1996 to December 2005 that met the following criteria: dementia (all-cause, Alzheimer disease or vascular dementia) as the outcome; longitudinal cohort study; study population broadly reflective of Canadian demographics; and genetic risk factors and general risk factors (e.g., hypertension, education, occupation and chemical exposure) identified. We graded the strength of evidence using the criteria of the Canadian Task Force on Preventive Health Care.

Results

Of 3424 articles on potentially modifiable risk factors for dementia, 1719 met our inclusion criteria; 60 were deemed to be of good or fair quality. Of 1721 articles on genetic risk factors, 62 that met our inclusion criteria were deemed to be of good or fair quality. On the basis of evidence from these articles, we made recommendations for the risk assessment and primary prevention of Alzheimer disease. For the primary prevention of Alzheimer''s disease, there is good evidence for controlling vascular risk factors, especially hypertension (grade A), and weak or insufficient evidence for manipulation of lifestyle factors and prescribing of medications (grade C). There is good evidence to avoid estrogens and high-dose (> 400 IU/d) of vitamin E for this purpose (grade E). Genetic counselling and testing may be offered to at-risk individuals with an apparent autosomal dominant inheritance (grade B). Screening for the apolipoprotein E genotype in asymptomatic individuals in the general population is not recommended (grade E).

Interpretation

Despite the personal and societal burden of dementia, our understanding of genetic predisposition to dementias and the contribution of other risk factors remains limited. More importantly, there are few data to explain the overall risks and benefits of prevention strategies or their impact of risk modification.

Articles to date in this series

• Chertkow H. Diagnosis and treatment of dementia: Introduction. Introducing a series based on the Third Canadian Consensus Conference on the Diagnosis and Treatment of Dementia. CMAJ 2008;178:316-21.

     

Identification of the major TG4 cross‐linking sites in the androgen‐dependent SVS I exclusively expressed in mouse seminal vesicle

SVS I was exclusively expressed in seminal vesicle in which the protein was immunolocalized primarily to the luminal epithelium of mucosal folds. The developmental profile of its mRNA expression was shown to be androgen‐dependent, manifesting a positive correlation with the animal's maturation. There are 43 glutamine and 43 lysine residues in one molecule of SVS I, which is one of the seven major monomer proteins tentatively assigned on reducing SDS–PAGE during the resolution of mouse seminal vesicle secretion. Based on the fact that SVS I‐deduced protein sequence consists of 796 amino acid residues, we produced 7 recombinant polypeptide fragments including residues 1–78/F1, residues 79–259/F2, residues 260–405/F3, residues 406–500/F4, residues 501–650/F5, residues 651–715/F6, and residues 716–796/F7, and measured the covalent incorporation of 5‐(biotinamido)pentylamine (BPNH₂) or biotin‐TVQQEL (A25 peptide) to each of F1‐to‐F7 by type 4 transglutaminase (TG₄) from the coagulating gland secretion. F2 was active to a greater extent than the other fragments during the BPNH₂‐glutamine incorporation, and a relatively low extent of A25‐lysine cross link was observed with all of the seven fragments. The MS analysis of BPNH₂‐F2 conjugate identified Q²³² and Q²⁵⁴ as the two major TG₄ cross‐linking sites. This was substantiated by the result that much less BPNH₂ was cross‐linked to any one of the three F2 mutants, including Q232G and Q254G obtained from single‐site mutation, and Q232G/Q254G from double‐site mutation. J. Cell. Biochem. 107: 899–907, 2009. © 2009 Wiley‐Liss, Inc.