Peptidoglycan and teichoic acid from Staphylococcus epidermidis stimulate human monocytes to release tumour necrosis factor-α, interleukin-1β and interleukin-6

Abstract Cytokines play a major role in the pathophysiology of septic shock. In this study, human periferal blood monocytes were stimulated with peptidoglycan and teichoic acid, purified from a strain of Staphylococcus epidermidis . Polymyxin B (PM-B) was added to avoid the effects of possible contamination with endotoxin. Tumour necrosis factor-α (TNF), interleukin-1β (IL-1), and interleukin-6 (IL-6) in the supernates were measured by enzyme-linked immunosorbent assays. Peptidoglycan and teichoic acid induced TNF, IL-1, and IL-6 in a concentration-dependent manner. Teichoic acid was a weaker inducer than peptidoglycan, especially for IL-1. Lipopolysaccharide from an E. coli strain was used as a control, being 100–1000 times more potent than peptidoglycan and teichoic acid.     

Use of the KlADH4 Promoter for Ethanol-Dependent Production of Recombinant Human Serum Albumin in Kluyveromyces lactis

KlADH4 is a gene of Kluyveromyces lactis encoding a mitochondrial alcohol dehydrogenase activity which is specifically induced by ethanol. The promoter of this gene was used for the expression of heterologous proteins in K. lactis, a very promising organism which can be used as an alternative host to Saccharomyces cerevisiae due to its good secretory properties. In this paper we report the ethanol-driven expression in K. lactis of the bacterial β-glucuronidase and of the human serum albumin (HSA) genes under the control of the KlADH4 promoter. In particular, we studied the extracellular production of recombinant HSA (rHSA) with integrative and replicative vectors and obtained a significant increase in the amount of the protein with multicopy vectors, showing that no limitation of KlADH4 trans-acting factors occurred in the cells. By deletion analysis of the promoter, we identified an element (UAS_E) which is sufficient for the induction of KlADH4 by ethanol and, when inserted in the respective promoters, allows ethanol-dependent activation of other yeast genes, such as PGK and LAC4. We also analyzed the effect of medium composition on cell growth and protein secretion. A clear improvement in the production of the recombinant protein was achieved by shifting from batch cultures (0.3 g/liter) to fed-batch cultures (1 g/liter) with ethanol as the preferred carbon source.     

Free brownian motion of individual lipid molecules in biomembranes

The mobility of phospolipids in free-standing and supported membranes was investigated on the level of individual molecules. For the analysis of trajectories a new statistical treatment was developed that permitted us to clearly distinguish different types of diffusional motion. A freely diffusing subfraction of lipids within supported membranes was identified. Its mobility was characterized by a mean lateral diffusion constant of D(supp) = 4.6 &mgr;m(2)/s. In comparison, the mobility of lipids embedded in "free-standing" planar membranes yielded an increase in the mean diffusion constant by a factor of 4.5, D(free) = 20.6 &mgr;m(2)/s. This increase is attributed to the ultrathin (</=1 nm) lubricating water layer between membranes and glass support.     

Adverse environmental conditions influence age-related innate immune responsiveness

Background-  

The innate immune system plays an important role in the recognition and induction of protective responses against infectious pathogens, whilst there is increasing evidence for a role in mediating chronic inflammatory diseases at older age. Despite indications that environmental conditions can influence the senescence process of the adaptive immune system, it is not known whether the same holds true for the innate immune system. Therefore we studied whether age-related innate immune responses are similar or differ between populations living under very diverse environmental conditions.     

Single amino acid supplementation in aminoacidopathies: a systematic review

Aminoacidopathies are a group of rare and diverse disorders, caused by the deficiency of an enzyme or transporter involved in amino acid metabolism. For most aminoacidopathies, dietary management is the mainstay of treatment. Such treatment includes severe natural protein restriction, combined with protein substitution with all amino acids except the amino acids prior to the metabolic block and enriched with the amino acid that has become essential by the enzymatic defect. For some aminoacidopathies, supplementation of one or two amino acids, that have not become essential by the enzymatic defect, has been suggested. This so-called single amino acid supplementation can serve different treatment objectives, but evidence is limited. The aim of the present article is to provide a systematic review on the reasons for applications of single amino acid supplementation in aminoacidopathies treated with natural protein restriction and synthetic amino acid mixtures.     

Regenerative potential of human muscle stem cells in chronic inflammation

Introduction

Chronic inflammation is a profound systemic modification of the cellular microenvironment which could affect survival, repair and maintenance of muscle stem cells. The aim of this study was to define the role of chronic inflammation on the regenerative potential of satellite cells in human muscle.

Methods

As a model for chronic inflammation, 11 patients suffering from rheumatoid arthritis (RA) were included together with 16 patients with osteoarthritis (OA) as controls. The mean age of both groups was 64 years, with more females in the RA group compared to the OA group. During elective knee replacement surgery, a muscle biopsy was taken from the distal musculus vastus medialis. Cell populations from four RA and eight OA patients were used for extensive phenotyping because these cell populations showed no spontaneous differentiation and myogenic purity greater than 75% after explantation.

Results

After mononuclear cell explantation, myogenic purity, viability, proliferation index, number of colonies, myogenic colonies, growth speed, maximum number of population doublings and fusion index were not different between RA and OA patients. Furthermore, the expression of proteins involved in replicative and stress-induced premature senescence and apoptosis, including p16, p21, p53, hTERT and cleaved caspase-3, was not different between RA and OA patients. Mean telomere length was shorter in the RA group compared to the OA group.

Conclusions

In the present study we found evidence that chronic inflammation in RA does not affect the in vitro regenerative potential of human satellite cells. Identification of mechanisms influencing muscle regeneration by modulation of its microenvironment may, therefore, be more appropriate.     

Toxicity,interstrand cross-links and DNA fragmentation induced by ‘activated’ cyclophosphamide in yeast: Comparative studies on 4-hydroperoxy-cyclophosphamide,its monofunctional analogon,acrolein, phosphoramide mustard,and nor-nitrogen mustard

Activated cyclophosphamide (CP) is known to achieve its cytotoxic and alkylating capacity upon spontaneous hydrolytic breakdown of the oxazaphosphorine ring structure. Treatment of yeast cells with the chemically activated form of CP (4-hydroperoxy-CP, 4-OOH-CP) and with several potentially toxic cleavage products revealed that cytotoxicity is closely linked to the formation of DNA interstrand cross-links and to DNA fragmentation. While this holds true for 4-OOH-CP and its bifunctional alkylating breakdown products, phosphoramide mustard (PM) and nor-nitrogen mustard (NNM), equimolar concentrations of acrolein and the monofunctional analogon of activated CP were inactive. NNM, the ultimate cleavage product within the successive degradation of the oxazaphosphorine structure was five times more toxic than 4-OOH-CP, whereas the cytotoxic action of PM was only slightly enhanced. The high cytotoxicity of NNM was matched by its ability to induce DNA interstrand cross-links: at concentrations and treatment times producing equal cell killing, 4-OOH-CP and NNM produced the same extent of cross-linking and DNA fragmentation. Biochemical potency of NNM is in contrast to data found with the NBP colorimetric assay which suggest that NNM loses its alkylating activity at neutral pH. 4-OOH-CP and PM are much more stable than predicted from half-life measurements performed via the NBP colorimetric assay: they retain a considerable fraction of their cytotoxic and cross-linking activity in spite of a 12-h preincubation at pH 7 and 36°C.     

Historical applications of induced sterilisation in field populations of mosquitoes

Research on sterile mosquito technology from 1955 to the 1980s provided a substantial body of knowledge on propagation and release of sterile mosquitoes. Radiation sterilisation and chemosterilisation have been used effectively to induce dominant lethality and thereby sterilise important mosquito vectors in the laboratory. Experimental releases of chemosterilised males provided complete control of Anopheles albimanus in a small breeding population (14-15 sq km) in El Salvador. Releases of radiation sterilised males failed to control either Aedes aegypti or Anopheles quadrimaculatus in the USA. Releases of radiation-sterilised and chemosterilised male Culex quinquefasciatus in the USA and India were successful in some instances. Development of genetic sexing systems for Anopheles and improved physical separation methods for Culex have made it possible to rear and release males almost exclusively (> 99%) minimizing the release of potential vectors, the females. Factors that affected efficacy in some field programmes included reduction of competitiveness by radiation, immigration of fertilized females from outside the release zones, and inability of laboratory-bred males to perform in the wild. Despite significant progress, institutional commitments to carry the process further were generally lacking in the late 1970s and until recently. Now, with renewed interest and support for further assessment of this technology, this paper summarizes the current knowledge base, prioritizes some areas of investigation, and challenges scientists and administrators to maintain an awareness of progress, remain realistic about the interpretation of new findings, and make decisions about the sterile insect technique on the basis of informed scientific documentation. Areas recommended for priority research status include the establishment of genetic sexing mechanisms that can be transferred to other mosquito species, re-examination of radiation sterilisation, aerial release technology and mass rearing.     

The PagN protein of <Emphasis Type="Italic">Salmonella enterica</Emphasis> serovar Typhimurium is an adhesin and invasin

Background  

The pagN gene of Salmonella enterica serovar Typhimurium is a PhoP-regulated gene that is up-regulated during growth within macrophages and in vivo in murine models of infection. The PagN protein displays similarity to the Hek and Tia invasins/adhesins of Escherichia coli. Thus far no function has been ascribed to the PagN protein.