Microbial production and application of sophorolipids

Sophorolipids are surface-active compounds synthesized by a selected number of yeast species. They have been known for over 40 years, but because of growing environmental awareness, they recently regained attention as biosurfactants due to their biodegradability, low ecotoxicity, and production based on renewable resources. In this paper, an overview is given of the producing yeast strains and various aspects of fermentative sophorolipid production. Also, the biochemical pathways and regulatory mechanisms involved in sophorolipid biosynthesis are outlined. To conclude, a summary is given on possible applications of sophorolipids, either as native or modified molecules.     

Gene delivery by cationic lipid vectors: overcoming cellular barriers

Non-viral vectors such as cationic lipids are capable of delivering nucleic acids, including genes, siRNA or antisense RNA into cells, thus potentially resulting in their functional expression. These vectors are considered as an attractive alternative for virus-based delivery systems, which may suffer from immunological and mutational hazards. However, the efficiency of cationic-mediated gene delivery, although often sufficient for cell biological purposes, runs seriously short from a therapeutics point of view, as realizing this objective requires a higher level of transfection than attained thus far. To develop strategies for improvement, there is not so much a need for novel delivery systems. Rather, better insight is needed into the mechanism of delivery, including lipoplex–cell surface interaction, route of internalization and concomitant escape of DNA/RNA into the cytosol, and transport into the nucleus. Current work indicates that a major obstacle involves the relative inefficient destabilization of membrane-bounded compartments in which lipoplexes reside after their internalization by the cell. Such an activity requires the capacity of lipoplexes of undergoing polymorphic transitions such as a membrane destabilizing hexagonal phase, while cellular components may aid in this process. A consequence of the latter notion is that for development of a novel generation of delivery devices, entry pathways have to be triggered by specific targeting to select delivery into intracellular compartments which are most susceptible to lipoplex-induced destabilization, thereby allowing the most efficient release of DNA, a minimal requirement for optimizing non-viral vector-mediated transfection. Dedicated to Prof. K. Arnold on the occasion of his 65th birthday.     

Cholesteryl ester transfer protein and hyperalphalipoproteinemia in Caucasians

It is unclear whether cholesteryl ester transfer protein (CETP) contributes to high density lipoprotein cholesterol (HDL-C) levels in hyperalphalipoproteinemia (HALP) in Caucasians. Moreover, even less is known about the effects of hereditary CETP deficiency in non-Japanese. We studied 95 unrelated Caucasian individuals with HALP. No correlations between CETP concentration or activity and HDL-C were identified. Screening for CETP gene defects led to the identification of heterozygosity for a novel splice site mutation in one individual. Twenty-five heterozygotes for this mutation showed reduced CETP concentration (-40%) and activity (-50%) and a 35% increase of HDL-C compared with family controls. The heterozygotes presented with an isolated high HDL-C, whereas the remaining subjects exhibited a typical high HDL-C/low-triglyceride phenotype. The increase of HDL-C in the CETP-deficient heterozygotes was primarily attributable to increased high density lipoprotein containing apolipoprotein A-I and A-II (LpAI:AII) levels, contrasting with an increase in both high density lipoprotein containing apolipoprotein A-I only and LpAI:AII in the other group. This study suggests the absence of a relationship between CETP and HDL-C levels in Caucasians with HALP. The data furthermore indicate that genetic CETP deficiency is rare among Caucasians and that this disorder presents with a phenotype that is different from that of subjects with HALP who have no mutation in the CETP gene.     

Comparison of naturally acquired antibody responses against the C-terminal processing products of Plasmodium vivax Merozoite Surface Protein-1 under low transmission and unstable malaria conditions in Sri Lanka

We report here, for the first time, a comparison of naturally acquired antibody responses to the 42 and 19 kDa C-terminal processing products of Plasmodium vivax Merozoite Surface Protein-1 assayed by ELISA using p42 and p19 baculovirus-derived recombinant proteins, respectively. Test populations comprised patients with microscopy confirmed acute P. vivax infections from two regions endemic for vivax malaria where low transmission and unstable malaria conditions prevail, and a non-endemic urban area, in Sri Lanka. The antibody prevalence to the two proteins, both at the individual and population levels, tend to respond more to p42 than to p19 in all test areas, where >14% of individuals preferentially recognized p42, compared with <2% for p19. In patients with no previous exposure to malaria, 21% preferentially recognized p42, whereas none exclusively recognized p19. A significantly lower prevalence of anti-p19 IgM, but not anti-p42 IgM, was observed among residents from endemic areas compared with their non-endemic counterparts. Individuals from both endemic areas produced significantly less anti-p19 IgM compared with anti-p42 IgM. IgG1 was the predominant IgG isotype for both antigens in all individuals. With increasing exposure to malaria in both endemic areas, anti-p19 antibody responses were dominated by the functionally important IgG1 and IgG3 isotypes, with a concurrent reduction in IgM that was lacking in the non-endemic residents. This antibody switch was also reflected for PvAMA-1 as we previously reported with the identical battery of sera. In contrast, the antibody switch for p42 was restricted to endemic residents with more extensive exposure. These results suggest that an IgM-dominated antibody response against the p42 polymorphic region in endemic residents may interfere with the development of an IgG-dominated "protective" isotype shift to p19, that may complicate vaccine development.     

Pulmonary expression of voltage-gated calcium channels: special reference to sensory airway receptors

Studying depolarisation induced calcium entry in our recently developed in situ lung slice model for molecular live cell imaging of selectively visualised pulmonary neuroepithelial bodies (NEBs), exemplified the need for information on the localisation of voltage-gated calcium channels (Ca(v)) in lungs in general, and related to sensory airway receptors more specifically. The present study therefore aimed at identifying the expression pattern of all major classes and subtypes of Ca(v) channels, using multiple immunostaining of rat lung cryosections. Ca(v) channel antibodies were combined with antibodies that selectively label NEBs, nerve fibre populations, smooth muscle, endothelium and Clara cells. Ca(v)2.1 (P/Q-type) was the only Ca(v) channel expressed in NEB cell membranes, and appeared to be restricted to the apical membrane of the slender NEB cell processes that reach the airway lumen. Subpopulations of the vagal but not the spinal sensory nerve fibres that contact NEBs showed immunoreactivity (IR) for Ca(v)1.2 (L-type) and Ca(v)2.1. Ca(v)2.3 (R-type) was selectively expressed by the so-called Clara-like cells that cover NEBs only, and appears to be a unique marker to discriminate this epithelial cell type from the much more extensive group of Clara cells in rat airways. The laminar nerve endings of smooth muscle-associated airway receptors (SMARs) revealed IR for both Ca(v)2.1 and Ca(v)2.2 (N-type). More generally, Ca(v)1.2 was seen to be expressed in vascular smooth muscle, Ca(v)2.3 and Ca(v)3.1 (T-type) in bronchial smooth muscle, Ca(v)3.1 and Ca(v)3.2 (T-type) in endothelial cells, and Ca(v)1.3 (L-type) in a limited number of epithelial cells. In conclusion, the present immunocytochemical study has demonstrated that the various subtypes of Ca(v) channels have distinct expression patterns in rat lungs. Special focus on morphologically/neurochemically characterised sensory airway receptors learned us that both NEBs and SMARs present Ca(v) channels. Knowledge of the identification and localisation of Ca(v) channels in airway receptors and surrounding tissues provides a solid basis for interpretation of the calcium mediated activation studied in our ex vivo lung slice model.     

Chaperoning Anfinsen: the steric foldases

Some proteins are so much resistant to proteolysis and unfolding that they violate folding rules shared by the vast majority of proteins. These unusual proteins manage to fold into an active native conformation that is thermodynamically at best marginally, but often even less stable than the unfolded state. A huge energetic barrier traps these proteins kinetically in the folded state. The drawback of this situation is the need for a specialized chaperone that adds steric information to the proteins to cross this barrier on the folding pathway. Until now, our knowledge of these intriguing chaperones was restricted to the prodomains of secreted proteases, which function intramolecularly. Recent research has added more examples, which now include the membrane-anchored lipase-specific foldase and the pilus subunit specific chaperone, both acting intermolecularly. The case of the pilin chaperone is somewhat deviant in that steric information is definitely provided, but the pilus subunit adopts a thermodynamically favourable stable conformation.     

Induction of heme oxygenase-1 and heat shock protein 70 in rat hepatocytes: The role of calcium signaling

Stress response genes including heat shock proteins are induced under a variety of conditions to confer cellular protection. This study investigated the role of calcium signaling in the induction of two stress response genes, heme oxygenase-1/hsp32 and hsp70, in isolated rat hepatocytes. Both genes were induced by cellular glutathione depletion. This induction could be inhibited by BAPTA-AM. Culturing in a calcium-free medium prevented the induction of hsp70 gene expression after glutathione depletion without affecting heme oxygenase-1 gene expression. Thapsigargin increased the gene expression of heme oxygenase-1 but not that of hsp70. Thapsigargin-induced heme oxygenase-1 induction was completely inhibited by BAPTA-AM. Incubation with the Ca²⁺-ionophore A23187 augmented heme oxygenase-1 (two-fold) and hsp70 (5.2-fold) mRNA levels. Our data suggests a significant role of Ca²⁺-dependent pathways in the induction of the two stress genes. An increase in the cytoplasmic Ca²⁺ activity seems to play a key role in the cascade of signaling leading to the induction of the two genes. However, the source of Ca²⁺ that fluxes into the cytoplasm seems to be different. Our data provides evidence for a compartmentalization of calcium fluxes, i.e. the Ca²⁺ flux from intracellular stores (e.g. the endoplasmic reticulum) plays a major role in the induction of heme oxygenase-1. By contrast, Ca²⁺ flux from the extracellular medium seems to be a mechanism initiating the cellular signaling cascade leading to hsp70 gene induction.     

Comparative analysis of genome maintenance genes in naked mole rat,mouse, and human

Genome maintenance (GM) is an essential defense system against aging and cancer, as both are characterized by increased genome instability. Here, we compared the copy number variation and mutation rate of 518 GM‐associated genes in the naked mole rat (NMR), mouse, and human genomes. GM genes appeared to be strongly conserved, with copy number variation in only four genes. Interestingly, we found NMR to have a higher copy number of CEBPG, a regulator of DNA repair, and TINF2, a protector of telomere integrity. NMR, as well as human, was also found to have a lower rate of germline nucleotide substitution than the mouse. Together, the data suggest that the long‐lived NMR, as well as human, has more robust GM than mouse and identifies new targets for the analysis of the exceptional longevity of the NMR.     

Vegetarianism,female gender and increasing age,but not <Emphasis Type="Italic">CNDP1</Emphasis> genotype,are associated with reduced muscle carnosine levels in humans

Carnosine is found in high concentrations in skeletal muscles, where it is involved in several physiological functions. The muscle carnosine content measured within a population can vary by a factor 4. The aim of this study was to further characterize suggested determinants of the muscle carnosine content (diet, gender and age) and to identify new determinants (plasma carnosinase activity and testosterone). We investigated a group of 149 healthy subjects, which consisted of 94 men (12 vegetarians) and 55 women. Muscle carnosine was quantified in M. soleus, gastrocnemius and tibialis anterior using magnetic resonance proton spectroscopy and blood samples were collected to determine CNDP1 genotype, plasma carnosinase activity and testosterone concentrations. Compared to women, men have 36, 28 and 82% higher carnosine concentrations in M. soleus, gastrocnemius and tibialis anterior muscle, respectively, whereas circulating testosterone concentrations were unrelated to muscle carnosine levels in healthy men. The carnosine content of the M. soleus is negatively related to the subjects’ age. Vegetarians have a lower carnosine content of 26% in gastrocnemius compared to omnivores. In contrast, there is no difference in muscle carnosine content between omnivores with a high or low ingestion of β-alanine. Muscle carnosine levels are not related to the polymorphism of the CNDP1 gene or to the enzymatic activity of the plasma carnosinase. In conclusion, neither CNDP1 genotype nor the normal variation in circulating testosterone levels affects the muscular carnosine content, whereas vegetarianism, female gender and increasing age are the factors associated with reduced muscle carnosine stores.