Knockout of the regulatory site of 3-ketoacyl-ACP synthase III enhances short- and medium-chain acyl-ACP synthesis

3-ketoacyl-acyl carrier protein synthase (KAS) III catalyses the first condensing step of the fatty acid synthase (FAS) type II reaction in plants and bacteria, using acetyl CoA and malonyl-acyl carrier protein (ACP) as substrates. Enzymatic characterization of recombinant KAS III from Cuphea wrightii embryo shows that this enzyme is strongly inhibited by medium-chain acyl-ACP end products of the FAS reaction, i.e. inhibition by lauroyl-ACP was uncompetitive towards acetyl CoA and non-competitive with regard to malonyl-ACP. This indicated a distinct attachment site for regulatory acyl-ACPs. Based on alignment of primary structures of various KAS IIIs and 3-ketoacyl CoA synthases, we suspected the motif G290NTSAAS296 to be responsible for binding of regulatory acyl-ACPs. Deletion of the tetrapeptide G290NTS293 led to a change of secondary structure and complete loss of KAS III condensing activity. Exchange of asparagine291 to aspartate, alanine294 to serine and alanine295 to proline, however, produced mutant enzymes with slightly reduced condensing activity, yet with insensitivity towards acyl-ACPs. To assess the potential of unregulated KAS III as tool in oil production, we designed in vitro experiments employing FAS preparations from medium-chain fatty acid-producing Cuphea lanceolata seeds and long-chain fatty acid-producing rape seeds, each supplemented with a fivefold excess of the N291D KAS III mutant. High amounts of short-chain acyl-ACPs in the case of C. lanceolata, and of medium-chain acyl-ACPs in the case of rape seed preparations, were obtained. This approach targets regulation and offers new possibilities to derive transgenic or non-transgenic plants for production of seed oils with new qualities.     

Survival of Vibrio fluvialis in seawater under starvation conditions

The viability of Vibrio fluvialis in seawater microcosms, with and without sediment was investigated. The strain survived as culturable bacteria for at least 1 year and the expression of its virulence factors was maintained. In microcosms containing sediment Vibrio fluvialis was more stable. Viable but nonculturable (VBNC) cells of Vibrio fluvialis were able to resuscitate to the culturable state up to 6 years of incubation in marine sediment. These cells recuperate their initial biochemical characteristics after 3 months of incubation in marine broth. Amplified 16S ribosomal DNA (rDNA) restriction analysis (ARDRA) was used to confirm that it is the same strain of Vibrio fluvialis which resists in all microcosms during a long period of time.     

The DEAD-box RNA helicase Ded1p affects and accumulates in Saccharomyces cerevisiae P-bodies

Recent results suggest that cytoplasmic mRNAs can form translationally repressed messenger ribonucleoprotein particles (mRNPs) capable of decapping and degradation, or accumulation into cytoplasmic processing bodies (P-bodies), which can function as sites of mRNA storage. The proteins that function in transitions between the translationally repressed mRNPs that accumulate in P-bodies and mRNPs engaged in translation are largely unknown. Herein, we demonstrate that the yeast translation initiation factor Ded1p can localize to P-bodies. Moreover, depletion of Ded1p leads to defects in P-body formation. Overexpression of Ded1p results in increased size and number of P-bodies and inhibition of growth in a manner partially suppressed by loss of Pat1p, Dhh1p, or Lsm1p. Mutations that inactivate the ATPase activity of Ded1p increase the overexpression growth inhibition of Ded1p and prevent Ded1p from localizing in P-bodies. Combined with earlier work showing Ded1p can have a positive effect on translation, these results suggest that Ded1p is a bifunctional protein that can affect both translation initiation and P-body formation.     

Barley γ3-hordein: Glycosylation at an atypical site,disulfide bridge analysis,and reactivity with IgE from patients allergic to wheat

Post translational modifications of a seed storage protein, barley γ3-hordein, were determined using immunochemical and mass spectrometry methods. IgE reactivity towards this protein was measured using sera from patients diagnosed with allergies to wheat. N-glycosylation was found at an atypical Asn-Leu-Cys site. The observed glycan contains xylose. This indicates that at least some γ3-hordein molecules trafficked through the Golgi apparatus. Disulfide bridges in native γ3-hordein were almost the same as those found in wheat γ46-gliadin, except the bridge involving the cysteine included in the glycosylation site. IgE reacted more strongly towards the recombinant than the natural γ3-hordein protein. IgE binding to γ3-hordein increased when the protein sample was reduced. Glycosylation and disulfide bridges therefore decrease epitope accessibility. Thus the IgE from patients sensitized to wheat cross-react with γ3-hordein due to sequence homology with wheat allergens rather than through shared carbohydrate determinants.     

Stability of serum-free and purified baculovirus stocks under various storage conditions

In a context of large-scale production of baculoviruses in serum-free media for use as gene delivery vectors, the stability of these viruses has become an important factor. The development of robust processes heavily relies on baculovirus stock stability. In the present work, we studied over a period of 300 days the stability of baculovirus vectors produced in serum-free media stored at 4, -20, or -80 degrees C or in liquid nitrogen. The viral stocks investigated were either crude baculovirus supernatant, baculovirus supernatant concentrated 10 times and diafiltered against fresh serum-free media by tangential flow filtration, or baculovirus purified by size exclusion chromatography. The results showed that baculovirus supernatant and diafiltered concentrate stored at 4 degrees C underwent a progressive loss of infectivity after a period of 100 and 50 days of storage, respectively. Aggregation has been recognized as the probable mechanism for the loss of infectivity. Baculovirus stocks were unstable at -20 degrees C, whereas in liquid nitrogen they retained infectivity after successive freeze thaw cycles. Concentration and diafiltration of baculovirus supernatant prior to storing at -80 degrees C contributed to improving viral stock stability over time. Glycerol as well as DMSO and sucrose have proven to be equally effective as additives to maintain the purified baculovirus stability after storage at -80 degrees C or in liquid nitrogen.     

Characterization of yeastolate fractions that promote insect cell growth and recombinant protein production

Yeastolate is effective in promoting growth of insect cell and enhancing production of recombinant protein, thus it is a key component in formulating serum-free medium for insect cell culture. However, yeastolate is a complex mixture and identification of the constituents responsible for cell growth promotion has not yet been achieved. This study used sequential ethanol precipitation to fractionate yeastolate ultrafiltrate (YUF) into six fractions (F1–F6). Fractions were characterized and evaluated for their growth promoting activities. Fraction F1 was obtained by 65% ethanol precipitation. When supplemented to IPL-41 medium at a concentration of 1 g L⁻¹, fraction F1 showed 71% Sf-9 cell growth improvement and 22% β-galactosidase production enhancement over YUF (at 1 g L⁻¹ in IPL-41 medium). However, the superiority of F1 over YUF on promoting cell growth gradually diminished as its concentration in IPL-41 medium increased. At 4 g L⁻¹, the relative activity of F1 was 93% whereas YUF was 100% at the same concentration. At 1 g L⁻¹, four other fractions (F2–F5) precipitated with higher ethanol concentrations and F6, the final supernatant, showed growth promoting activities ranging from 32 to 80% as compared to YUF (100%). Interestingly, a synergistic effect on promoting cell growth was observed when F6 was supplemented in IPL-41 medium in presence of high concentrations of F1 (>3 g L⁻¹). The results suggest that ethanol precipitation was a practical method to fractionate growth-promoting components from YUF, but more than one components contributed to the optimum growth of Sf-9 cells. Further fractionation, isolation and identification of individual active components would be needed to better understand the role of these components on the cell metabolism.     

Proteomic characterization of lipid rafts markers from the rat intestinal brush border

To assess intestinal lipid rafts functions through the characterization of their protein markers, we have isolated lipid rafts of rat mucosa either from the total membrane or purified brush-border membrane (BBM) by sucrose gradient fractionation after detergent treatment. In both membrane preparations, the floating fractions (4-5) were enriched in cholesterol, ganglioside GM1, and N aminopeptidase (NAP) known as intestinal lipid rafts markers. Based on MALDI-TOF/MS identification and simultaneous detection by immunoblotting, 12 proteins from BBM cleared from contaminants were selected as rafts markers. These proteins include several signaling/trafficking proteins belonging to the G protein family and the annexins as well as GPI-anchored proteins. Remarkably GP2, previously described as the pancreatic granule GPI-anchored protein, was found in intestinal lipid rafts. The proteomic strategy assayed on the intestine leads to the characterization of known (NAP, alkaline phosphatase, dipeptidyl aminopeptidase, annexin II, and galectin-4) and new (GP2, annexin IV, XIIIb, Galpha(q), Galpha(11), glutamate receptor, and GPCR 7) lipid rafts markers. Together our results indicate that some digestive enzymes, trafficking and signaling proteins may be functionally distributed in the intestine lipid rafts.     

Masson’s tumor of the kidney: a case report

Background

Intravascular papillary endothelial hyperplasia (known also as Masson’s tumor) is a benign vascular lesion that commonly occurs in the skin and is rarely found in solid organs, especially in the kidney. In what follows, we will look into the first case of an unexpectedly diagnosed Masson’s tumor of the kidney presenting as a suspicious renal cyst.

Case presentation

A 61-year-old Arab man presented with a left renal cyst, incidentally revealed by ultrasonography. The laboratory values were unremarkable. Computed tomography and magnetic resonance imaging demonstrated a 38 mm left renal midportion Bosniak IV cyst. Our patient underwent a radical nephrectomy. Histopathology revealed the diagnosis of intravascular papillary endothelial hyperplasia. There was no recurrence detected after 9 years of follow-up.

Conclusions

Renal intravascular papillary endothelial hyperplasia is a rare benign tumor which can mimic a suspicious renal mass on radiological findings. Thus, this entity should be considered more often in the thick of the diagnostic possibilities in order to avoid unnecessary nephrectomies.

     

Critical assessment of current adeno-associated viral vector production and quantification methods

Adeno-associated viral vectors have emerged as one of the most studied vectors for gene therapy. Numerous production methods have been described, each with its advantages and disadvantages. A challenge in assessing the current state of the art exists in comparing yields from one production system to the next due to the wide variety of quantification techniques. In this review, AAV vector production methods are summarized and the yields of the different processes are standardized to the number of harvested cells. Titers are further streamlined into five categories: transduction units, enhanced transduction units, infectious particles, DNase-resistant particles and total particles, and the importance of each type of measure is discussed.