The involucrin genes of the mouse and the rat: study of their shared repeats

The involucrin genes of the mouse (Mus musculus) and the rat (Rattus norvegicus) have been cloned and sequenced. The coding region of each gene contains, at site P, a segment of repeats homologous to that of other nonanthropoid mammals. In contrast to the repeats of species belonging to different mammalian orders, many individual repeats of the mouse and the rat can be matched. Both before and after the divergence of the two species, these repeats have been the site of systematic alterations in nucleotide sequence. One of the alterations is the correction of nucleotides of one repeat by those of another. Corrected nucleotides may be closely linked to flanking nucleotides that are uncorrected; the systematic correction process therefore appears to be due to gene conversion. There is a stretch of 18 reiterated CAGs in the segment of repeats of the Mus gene; most of these reiterations were introduced recently, supporting the idea that the gene was generated originally from poly CAG. An antiserum to a synthetic peptide encoded by the segment of repeats of the Mus gene reveals differentiation- specific expression of the gene in the epidermis.      

Membrane fatty acid composition and endothelial cell functional properties

In order to study the influence of endothelial cell fatty acid composition on various membrane related parameters, several in vitro methods were developed for manipulating the fatty acid content of human endothelial cell membranes. Changes in membrane fatty acid profile were induced by using fatty acid modified lipoproteins or free fatty acids. The largest changes in endothelial fatty acid composition were obtained by culturing the cells in media supplemented with specific free fatty acids. An increase in arachidonic acid content of endothelial phospholipids was induced by supplementation with saturated fatty acids or with arachidonic acid itself. A decrease in arachidonic acid content was obtained by supplementation with other unsaturated fatty acids. Under the experimental conditions used endothelial cells showed a low desaturase activity and a high elongase activity. Considerable alterations in membrane fatty acid composition did not greatly influence certain membrane related parameters such as polymorphonuclear leukocyte adherence and endothelial cell procoagulant activity. In general, for fatty acid modified endothelial cells an association between endogenous arachidonic acid content and total production of eicosanoids was found. This study demonstrates that considerable changes in membrane fatty acid profile affect endothelial cell arachidonic acid metabolism, but it also illustrates homeostasis at the level of endothelial cell functional activity.     

Heterologous gene expression in Lactococcus lactis subsp. lactis: synthesis, secretion, and processing of the Bacillus subtilis neutral protease.

The Bacillus subtilis nprE gene lacking its own promoter sequence was inserted in the lactococcal expression vector pMG36e. Upon introduction of the recombinant plasmid into Lactococcus lactis subsp. lactis strain MG1363, neutral protease activity could be visualized by the appearance of large clearing zones around colonies grown on milk agar plates. By measuring the activities of the neutral protease and the intracellular enzyme lactate dehydrogenase in culture supernatants and cell fractions, it was demonstrated that the neutral protease was actively secreted into the growth medium. This was corroborated by using the Western blot (immunoblot) technique, which showed the presence of the mature form of the neutral protease in the culture supernatant. On the basis of these results, it is concluded that the B. subtilis neutral protease gene was expressed in L. lactis and that the gene product was secreted into the growth medium and was apparently correctly processed to produced a biologically active protein. The secretion of this particular enzyme may be helpful in achieving accelerated cheese ripening.     

Development of a detection system for histidine decarboxylating lactic acid bacteria based on DNA probes, PCR and activity test

On the basis of the comparison of the nucleotide sequences of the histidine decarboxylase genes ( hdc A) of Lactobacillus 30A and Clostridium perfringens and the amino acid sequences of these histidine decarboxylases and those of Lactobacillus buchneri and Micrococcus , oligonucleotides unique to the hdc A genes were synthesized and used in PCR. All histidine-decarboxylating lactic acid bacteria gave a signal with primer set JV16HC/JV17HC in PCR. In addition to this primer set, CL1/CL2 and CL1/JV17HC were also useful for the detection of histamine-forming Leuconostoc œnos strains in PCR. The 150 base pair amplification product of the decarboxylating Leuc. œnos strain generated with primer set CL1/CL2 was sequenced. Alignment studies showed a high degree of relatedness among the hdc A gene products of Gram-positive bacteria.
The amplification products of the hdc A genes from Lact. buchneri and Leuc. œnos were used to serve as a DNA probe in hybridization studies. All histidine-decarboxylating lactic acid bacteria gave a hybridization signal with the DNA probes. In hybridization only one false-positive signal with a Lactobacillus lindneri strain was observed, which was anticipated to contain a truncated hdc A gene.
In addition to these DNA probe tests, a simple and reliable activity test is presented, which can be used during starter selection to test strains for histidine decarboxylase activity.     

Intradermally administered yellow fever vaccine at reduced dose induces a protective immune response: a randomized controlled non-inferiority trial

Background

Implementation of yellow fever vaccination is currently hampered by limited supply of vaccine. An alternative route of administration with reduced amounts of vaccine but without loss of vaccine efficacy would boost vaccination programmes.

Methods and Findings

A randomized, controlled, non-inferiority trial was conducted in a Dutch university center between August 2005 and February 2007. A total of 155 primary vaccinated and 20 previously vaccinated volunteers participated. Participants were randomly assigned in a 1∶1 ratio to receive intradermal (i.d.) vaccination with live attenuated yellow fever 17D vaccine at a reduced dose (1/5^th; 0·1 mL) or the conventional subcutaneous (s.c.) vaccination (0·5 mL). Antibody neutralization titers were determined at 2, 4 and 8 weeks and 1 year after vaccination by counting the reduction in virus-induced plaques in the presence of serial serum dilutions. Adverse events were documented in a 3-week dairy. Viraemia was measured 5 days after vaccination. From 2 weeks up to one year after vaccination, the maximum serum-dilution at which 80% of the virus plaques were neutralized, which indicates protection against yellow fever, did not differ between those given a reduced i.d. dose or standard s.c. dose of vaccine. In all cases the WHO standard of seroprotection (i.e. 80% virus neutralization) was reached (in 77/77 and 78/78, respectively). Similar results were found in the previously vaccinated individuals. Viraemia was detected in half of the primary vaccinated participants, which was not predictive of serological response. In revaccinees no viraemia was detected.

Conclusions

Intradermal administration of one fifth of the amount of yellow fever vaccine administered subcutaneously results in protective seroimmunity in all volunteers. Albeit this vaccination route should enable vaccination of five-times as many individuals at risk for disease, these results should now be confirmed in field studies in areas with potential yellow fever virus transmission to change vaccination policy.

Trial Registration

Nederlands Trial Register ISRCTN46326316     

The genetic influence on the cortical processing of experimental pain and the moderating effect of pain status

Background

Research suggests that the COMT Val¹⁵⁸Met, BDNF Val⁶⁶Met and OPRM1 A¹¹⁸G polymorphisms moderate the experience of pain. In order to obtain experimental confirmation and extension of findings, cortical processing of experimentally-induced pain was used.

Method

A sample of 78 individuals with chronic low back pain complaints and 37 healthy controls underwent EEG registration. Event-Related Potentials were measured in response to electrical nociceptive stimuli and moderation by COMT Val¹⁵⁸Met, BDNF Val⁶⁶Met and OPRM1 A¹¹⁸G polymorphisms was assessed.

Results

Genetic variation did not have a direct effect on cortical processing of experimental pain. However, genetic effects (COMT Val¹⁵⁸Met and BDNF Val⁶⁶Met) on experimental pain were moderated by the presence of chronic pain. In the presence of chronic pain, the COMT Met allele and the BDNF Met allele augmented cortical pain processing, whilst reducing pain processing in pain-free controls. No significant effects were found concerning the OPRM1 A¹¹⁸G polymorphism.

Conclusions

The current study suggests that chronic experience of pain enhances genetic sensitivity to experimentally induced mildly painful stimuli, possibly through a process of epigenetic modification.     

Cost-effective HRMA pre-sequence typing of clone libraries; application to phage display selection

Background  

Methodologies like phage display selection, in vitro mutagenesis and the determination of allelic expression differences include steps where large numbers of clones need to be compared and characterised. In the current study we show that high-resolution melt curve analysis (HRMA) is a simple, cost-saving tool to quickly study clonal variation without prior nucleotide sequence knowledge.