Stable expression of an active soluble recombinant form of human fucosyltransferase IX in Spodoptera frugiperda Sf9 cells

A secretory form of human α3-fucosyltransferase IX (sFUT9) was overexpressed in Spodoptera frugiperda (Sf9) insect cells using the stable expression vector pIB/V5-His-TOPO and the signal sequence of human interleukin 2 for efficient secretion. sFUT9 was active and its three potential N-glycosylation sites were occupied. sFUT9 efficiently fucosylated the type II acceptors Galbeta4GlcNAC-R and Fucalpha2Galbeta4GlcNAc-R (R = (CH2)3NHCO(CH2)5–NH-biotin) but not the corresponding sialylated acceptor, and only very poorly the type I (Galbeta3GlcNAc-R) related acceptors. sFUT9 showed a clear preference for glycoproteins containing type II acceptors, with values of 121, 113 and 110 microU/million cell for asialofetuin, erythropoietin and asialoerythropoietin, respectively, values approximately 11-fold higher than those obtained for the small acceptors.     

Production and purification of functional truncated soluble forms of human recombinant L1 cell adhesion glycoprotein from Spodoptera frugiperda Sf9 cells

L1 is a human cell adhesion glycoprotein involved in the development of the central nervous system that comprises six immunoglobulin-like domains (Ig1-Ig6), five fibronectin-type III (FN1-FN5) domains, a single transmembrane region and a cytoplasmic domain. It contains 20 potential N-glycosylation sites and is heavily glycosylated in a variety of cell types. In this work, seven truncated soluble forms including L1 ectodomain (L1/ECD) and Ig domains 5-6 (L1/Ig5-6) have been constructed by PCR and have been cloned, as well as the full-length form (L1), in the stable expression vector for insect cells pMIB/V5-His-TOPO. Spodoptera frugiperda Sf9 cell lines expressing the truncated forms have been obtained, and all proteins were successfully secreted. L1/ECD and L1/Ig5-6 were produced in shake flasks with productions of 3 and 32 mg/L on the third and fourth day of culture, respectively. When L1/Ig5-6 was produced for four days in 2L bioreactor 200 mg/L protein were recovered from the supernatants on the fourth day of culture. Affinity-purified L1/ECD and L1/Ig5-6 were immobilized on poly-d-lysine coated coverslips, and were shown to be active in inducing neurite outgrowth from human NT2N neurons. Therefore, correctly folded and functional truncated forms of human L1 have been produced in high amounts from insect cells using a stable expression system.     

Heterologous amplification and characterization of microsatellite markers in the Neotropical fish Leporinus friderici

Leporinus friderici, native to the Amazon Basin and popularly known as "piau-três-pintas", has great ecological and economic importance; it is widely fished and consumed throughout much of tropical South America. Knowledge of the genetic diversity of this native species is important to support management and conservation programs. We evaluated microsatellite loci amplification, using heterologous primers, in 31 individuals of L. friderici. These samples were collected from natural populations of the Araguaia River basin, in central Brazil, and the DNA was extracted from samples of muscle tissue. Eight loci were successfully analyzed. Six of them were polymorphic, and the number of alleles ranged from three to 10. Values of expected heterozygosities for these polymorphic loci ranged from 0.488 to 0.795. Exclusion probability (0.983), the identity probability (0.000073), and the mean genetic diversity values were high, showing that these microsatellite markers are suitable for assessing the genetic variability of L. friderici populations. There is a growing interest in studies that evaluate the genetic variability of natural populations for various purposes, such as conservation. Here, we showed that a viable alternative to the costly development of specific primers for fish populations is simply testing for heterologous amplification of microsatellite markers available from research on other species.     

Quantification of global transcription patterns in prokaryotes using spotted microarrays

We describe an analysis, applicable to any spotted microarray dataset produced using genomic DNA as a reference, that quantifies prokaryotic levels of mRNA on a genome-wide scale. Applying this to Mycobacterium tuberculosis, we validate the technique, show a correlation between level of expression and biological importance, define the complement of invariant genes and analyze absolute levels of expression by functional class to develop ways of understanding an organism's biology without comparison to another growth condition.     

An annotated catalogue of salivary gland transcripts in the adult female mosquito, Ædes ægypti*

Background

The number of completely sequenced plastid genomes available is growing rapidly. This array of sequences presents new opportunities to perform comParative analyses. In comParative studies, it is often useful to compare across wide phylogenetic spans and, within angiosperms, to include representatives from basally diverging lineages such as the genomes reported here: Nuphar advena (from a basal-most lineage) and Ranunculus macranthus (a basal eudicot). We report these two new plastid genome sequences and make comparisons (within angiosperms, seed plants, or all photosynthetic lineages) to evaluate features such as the status of ycf15 and ycf68 as protein coding genes, the distribution of simple sequence repeats (SSRs) and longer dispersed repeats (SDR), and patterns of nucleotide composition.

Results

The Nuphar [GenBank:NC_008788] and Ranunculus [GenBank:NC_008796] plastid genomes share characteristics of gene content and organization with many other chloroplast genomes. Like other plastid genomes, these genomes are A+T-rich, except for rRNA and tRNA genes. Detailed comparisons of Nuphar with Nymphaea, another Nymphaeaceae, show that more than two-thirds of these genomes exhibit at least 95% sequence identity and that most SSRs are shared. In broader comparisons, SSRs vary among genomes in s of abundance and length and most contain repeat motifs based on A and T nucleotides.

Conclusion

SSR and SDR abundance varies by genome and, for SSRs, is proportional to genome size. Long SDRs are rare in the genomes assessed. SSRs occur less frequently than predicted and, although the majority of the repeat motifs do include A and T nucleotides, the A+T bias in SSRs is less than that predicted from the underlying genomic nucleotide composition. In codon usage third positions show an A+T bias, however variation in codon usage does not correlate with differences in A+T-richness. Thus, although plastome nucleotide composition shows "A+T richness", an A+T bias is not apparent upon more in-depth analysis, at least in these aspects. The pattern of evolution in the sequences identified as ycf15 and ycf68 is not consistent with them being protein-coding genes. In fact, these regions show no evidence of sequence conservation beyond what is normal for non-coding regions of the IR.     

Ultrasound imaging in an experimental model of fatty liver disease and cirrhosis in rats

Background

Atypical scrapie was first identified in Norwegian sheep in 1998 and has subsequently been identified in many countries. Retrospective studies have identified cases predating the initial identification of this form of scrapie, and epidemiological studies have indicated that it does not conform to the behaviour of an infectious disease, giving rise to the hypothesis that it represents spontaneous disease. However, atypical scrapie isolates have been shown to be infectious experimentally, through intracerebral inoculation in transgenic mice and sheep. The first successful challenge of a sheep with 'field' atypical scrapie from an homologous donor sheep was reported in 2007.

Results

This study demonstrates that atypical scrapie has distinct clinical, pathological and biochemical characteristics which are maintained on transmission and sub-passage, and which are distinct from other strains of transmissible spongiform encephalopathies in the same host genotype.

Conclusions

Atypical scrapie is consistently transmissible within AHQ homozygous sheep, and the disease phenotype is preserved on sub-passage.     

Current genetic engineering strategies for the production of antihypertensive ACEI peptides

Hypertension is a major and highly prevalent risk factor for various diseases. Among the most frequently prescribed antihypertensive first-line drugs are synthetic angiotensin I-converting enzyme inhibitors (ACEI). However, since  their use in hypertension therapy has been linked to various side effects, interest in the application of food-derived ACEI peptides (ACEIp) as antihypertensive agents is rapidly growing. Although promising, the industrial production of ACEIp through conventional methods such as chemical synthesis or enzymatic hydrolysis of food proteins has been proven troublesome. We here provide an overview of current antihypertensive therapeutics, focusing on ACEI, and illustrate how biotechnology and bioengineering can overcome the limitations of ACEIp large-scale production. Latest advances in ACEIp research and current genetic engineering-based strategies for heterologous production of ACEIp (and precursors) are also presented. Cloning approaches include tandem repeats of single ACEIp, ACEIp fusion to proteins/polypeptides, joining multivariate ACEIp into bioactive polypeptides, and producing ACEIp-containing modified plant storage proteins. Although bacteria have been privileged ACEIp heterologous hosts, particularly when testing for new genetic engineering strategies, plants and microalgae-based platforms are now emerging. Besides being generally safer, cost-effective and scalable, these “pharming” platforms can perform therelevant posttranslational modifications and produce (and eventually deliver) biologically active protein/peptide-based antihypertensive medicines.     

Metabolic effect of telmisartan and losartan in hypertensive patients with metabolic syndrome

Background

Metabolic syndrome is a cluster of common cardiovascular risk factors that includes hypertension and insulin resistance. Hypertension and diabetes mellitus are frequent comorbidities and, like metabolic syndrome, increase the risk of cardiovascular events. Telmisartan, an antihypertensive agent with evidence of partial peroxisome proliferator-activated receptor activity-gamma (PPARγ) activity, may improve insulin sensitivity and lipid profile in patients with metabolic syndrome.

Methods

In a double-blind, parallel-group, randomized study, patients with World Health Organization criteria for metabolic syndrome received once-daily doses of telmisartan (80 mg, n = 20) or losartan (50 mg, n = 20) for 3 months. At baseline and end of treatment, fasting and postprandial plasma glucose, insulin sensitivity, glycosylated haemoglobin (HBA_1c) and 24-hour mean systolic and diastolic blood pressures were determined.

Results

Telmisartan, but not losartan, significantly (p < 0.05) reduced free plasma glucose, free plasma insulin, homeostasis model assessment of insulin resistance and HbA_ic. Following treatment, plasma glucose and insulin were reduced during the oral glucose tolerance test by telmisartan, but not by losartan. Telmisartan also significantly reduced 24-hour mean systolic blood pressure (p < 0.05) and diastolic blood pressure (p < 0.05) compared with losartan.

Conclusion

As well as providing superior 24-hour blood pressure control, telmisartan, unlike losartan, displayed insulin-sensitizing activity, which may be explained by its partial PPARγ activity.