Disruption of reducing pathways is not essential for efficient disulfide bond formation in the cytoplasm of <Emphasis Type="Italic">E. coli</Emphasis>

Background  

The formation of native disulfide bonds is a complex and essential post-translational modification for many proteins. The large scale production of these proteins can be difficult and depends on targeting the protein to a compartment in which disulfide bond formation naturally occurs, usually the endoplasmic reticulum of eukaryotes or the periplasm of prokaryotes. It is currently thought to be impossible to produce large amounts of disulfide bond containing protein in the cytoplasm of wild-type bacteria such as E. coli due to the presence of multiple pathways for their reduction.     

The 3120 +1G-->A splicing mutation in CFTR is common in Brazilian cystic fibrosis patients.

Cystic fibrosis patients from Rio de Janeiro, Brazil, were screened for mutations in exons 11 and 16 of the cystic fibrosis transmembrane conductance regulator gene (CFTR) by a nonradioactive single-stranded conformational polymorphism (SSCP) analysis technique. This procedure was used to evaluate the undefined mutations in one or both alleles of 64 cystic fibrosis patients. Unusual SSCP profiles were investigated further by sequence analysis. Two patients were shown to carry the G542X mutation (exon 11) and five had the splicing mutation 3120+1G-->A(intron 16), one of them being homozygous for the mutation. This is the first report of the 3120+ IG-->A mutation in Brazil. where it appears to be a frequent disease-associated molecular alteration in the CFTR gene.     

Modeling brewers' yeast flocculation

Flocculation of yeast cells occurs during the fermentation of beer. Partway through the fermentation the cells become flocculent and start to form flocs. If the environmental conditions, such as medium composition and fluid velocities in the tank, are optimal, the flocs will grow in size large enough to settle. After settling of the main part of the yeast the green beer is left, containing only a small amount of yeast necessary for rest conversions during the next process step, the lagering. The physical process of flocculation is a dynamic equilibrium of floc formation and floc breakup resulting in a bimodal size distribution containing single cells and flocs. The floc size distribution and the single cell amount were measured under the different conditions that occur during full scale fermentation. Influences on flocculation such as floc strength, specific power input, and total number of yeast cells in suspension were studied. A flocculation model was developed, and the measured data used for validation. Yeast floc formation can be described with the collision theory assuming a constant collision efficiency. The breakup of flocs appears to occur mainly via two mechanisms, the splitting of flocs and the erosion of yeast cells from the floc surface. The splitting rate determines the average floc size and the erosion rate determines the number of single cells. Regarding the size of the flocs with respect to the scale of turbulence, only the viscous subrange needs to be considered. With the model, the floc size distribution and the number of single cells can be predicted at a certain point during the fermentation. For this, the bond strength between the cells, the fractal dimension of the yeast, the specific power input in the tank and the number of yeast cells that are in suspension in the tank have to be known. Copyright 1998 John Wiley & Sons, Inc.     

Studies on the transposition rates of mobile genetic elements in a natural population of Drosophila melanogaster

In an isolated population of Drosophila melanogaster on Ishigaki Island the chromosomal distribution of several retrotransposons, including copia, 412, 297, 17.6, I, and jockey elements, was examined by in situ hybridization. In this population the cosmopolitan inversion, In(2L)t, is known to exist in high frequency. One major haplotype concerning the occupied sites of the transposable elements was identified in the In(2L)t-carrying chromosomes. This haplotype is suggested to be the ancestral one. The age of the inversion in this local population was estimated to be 1,400 generations. The transposition rates of these elements were estimated based on the age of the inversion and the number of the elements lost and gained. The excision rates were in the range from 9.13 x 10(-5) to 2.25 x 10(-4) per site per generation. They were similar each other in the copia-like elements as well as in the LINE-like elements. The rate was higher in the copia-like elements than in the LINE-like elements. Insertions occurred in the range from 6.79 x 10(-4) to 9.05 x 10(-4) per element per generation. It is herein shown that both insertions and excisions occurred at a significantly higher rate in this population than in the laboratory.      

Endogenous chloride channels of insect sf9 cells. Evidence for coordinated activity of small elementary channel units

The endogenous Cl- conductance of Spodoptera frugiperda (Sf9) cells was studied 20-35 h after plating out of either uninfected cells or cells infected by a baculovirus vector carrying the cloned beta-galactosidase gene (beta-Gal cells). With the cation Tris+ in the pipette and Na+ in the bath, the reversal potential of whole-cell currents was governed by the prevailing Cl- equilibrium potential and could be fitted by the Goldman-Hodgkin-Katz equation with similar permeabilities for uninfected and beta-Gal cells. In the frequency range 0.12 < f < 300 Hz, the power density spectrum of whole-cell Cl- currents could be fitted by three Lorentzians. Independent of membrane potential, >50% of the total variance of whole-cell current fluctuations was accounted for by the low frequency Lorentzian (fc = 0.40 +/- 0.03 Hz, n = 6). Single-Cl- channels showed complex gating kinetics with long lasting (seconds) openings interrupted by similar long closures. In the open state, channels exhibited fast burst-like closures. Since the patches normally contained more than a single channel, it was not possible to measure open and closed dwell-time distributions for comparing single-Cl- channel activity with the kinetic features of whole-cell currents. However, the power density spectrum of Cl- currents of cell-attached and excised outside-out patches contained both high and low frequency Lorentzian components, with the corner frequency of the slow component (fc = 0.40 +/- 0.02 Hz, n = 4) similar to that of whole-cell current fluctuations. Chloride channels exhibited multiple conductance states with similar Goldman-Hodgkin-Katz-type rectification. Single-channel permeabilities covered the range from approximately 0.6.10(-14) cm5/s to approximately 6.10(-14) cm3/s, corresponding to a limiting conductance (gamma 150/150) of approximately 3.5 pS and approximately 35 pS, respectively. All states reversed near the same membrane potential, and they exhibited similar halide ion selectivity, P1 > PCl approximately PBr. Accordingly, Cl- current amplitudes larger than current flow through the smallest channel unit resolved seem to result from simultaneous open/shut events of two or more channel units.     

High quality copy number and genotype data from FFPE samples using Molecular Inversion Probe (MIP) microarrays

Background

Novel tuberculosis (TB) vaccines recently tested in humans have been designed to boost immunity induced by the current vaccine, Mycobacterium bovis Bacille Calmette-Guérin (BCG). Because BCG vaccination is used extensively in infants, this population group is likely to be the first in which efficacy trials of new vaccines will be conducted. However, our understanding of the complexity of immunity to BCG in infants is inadequate, making interpretation of vaccine-induced immune responses difficult.

Methods

To better understand BCG-induced immunity, we performed gene expression profiling in five 10-week old infants routinely vaccinated with BCG at birth. RNA was extracted from 12 hour BCG-stimulated or purified protein derivative of tuberculin (PPD)-stimulated PBMC, isolated from neonatal blood collected 10 weeks after vaccination. RNA was hybridised to the Sentrix^® HumanRef-8 Expression BeadChip (Illumina) to measure expression of >16,000 genes.

Results

We found that ex vivo stimulation of PBMC with PPD and BCG induced largely similar gene expression profiles, except that BCG induced greater macrophage activation. The peroxisome proliferator-activated receptor (PPAR) signaling pathway, including PPAR-γ, involved in activation of the alternative, anti-inflammatory macrophage response was down-regulated following stimulation with both antigens. In contrast, up-regulation of genes associated with the classic, pro-inflammatory macrophage response was noted. Further analysis revealed a decrease in the expression of cell adhesion molecules (CAMs), including integrin alpha M (ITGAM), which is known to be important for entry of mycobacteria into the macrophage. Interestingly, more leukocyte genes were down-regulated than up-regulated.

Conclusion

Our results suggest that a combination of suppressed and up-regulated genes may be key in determining development of protective immunity to TB induced by vaccination with BCG.     

The many roles of small RNAs in leaf development

Leaf development involves many complex genetic interactions,signals between adjacent cells or between more distant tissues and consequent changes in cell fate.This review describes three stages in leaf development where regulation by small RNAs have been used to modulate gene expression patterns.     

Pre-expression of a sulfhydryl oxidase significantly increases the yields of eukaryotic disulfide bond containing proteins expressed in the cytoplasm of E.coli

Background  

Disulfide bonds are one of the most common post-translational modifications found in proteins. The production of proteins that contain native disulfide bonds is challenging, especially on a large scale. Either the protein needs to be targeted to the endoplasmic reticulum in eukaryotes or to the prokaryotic periplasm. These compartments that are specialised for disulfide bond formation have an active catalyst for their formation, along with catalysts for isomerization to the native state. We have recently shown that it is possible to produce large amounts of prokaryotic disulfide bond containing proteins in the cytoplasm of wild-type bacteria such as E. coli by the introduction of catalysts for both of these processes.     

A model of dengue fever

Background  

Dengue is a disease which is now endemic in more than 100 countries of Africa, America, Asia and the Western Pacific. It is transmitted to the man by mosquitoes (Aedes) and exists in two forms: Dengue Fever and Dengue Haemorrhagic Fever. The disease can be contracted by one of the four different viruses. Moreover, immunity is acquired only to the serotype contracted and a contact with a second serotype becomes more dangerous.