Putative Nanobacteria Represent Physiological Remnants and Culture By-Products of Normal Calcium Homeostasis

Putative living entities called nanobacteria (NB) are unusual for their small sizes (50–500 nm), pleomorphic nature, and accumulation of hydroxyapatite (HAP), and have been implicated in numerous diseases involving extraskeletal calcification. By adding precipitating ions to cell culture medium containing serum, mineral nanoparticles are generated that are morphologically and chemically identical to the so-called NB. These nanoparticles are shown here to be formed of amorphous mineral complexes containing calcium as well as other ions like carbonate, which then rapidly acquire phosphate, forming HAP. The main constituent proteins of serum-derived NB are albumin, fetuin-A, and apolipoprotein A1, but their involvement appears circumstantial since so-called NB from different body fluids harbor other proteins. Accordingly, by passage through various culture media, the protein composition of these particles can be modulated. Immunoblotting experiments reveal that antibodies deemed specific for NB react in fact with either albumin, fetuin-A, or both, indicating that previous studies using these reagents may have detected these serum proteins from the same as well as different species, with human tissue nanoparticles presumably absorbing bovine serum antigens from the culture medium. Both fetal bovine serum and human serum, used earlier by other investigators as sources of NB, paradoxically inhibit the formation of these entities, and this inhibition is trypsin-sensitive, indicating a role for proteins in this inhibitory process. Fetuin-A, and to a lesser degree albumin, inhibit nanoparticle formation, an inhibition that is overcome with time, ending with formation of the so-called NB. Together, these data demonstrate that NB are most likely formed by calcium or apatite crystallization inhibitors that are somehow overwhelmed by excess calcium or calcium phosphate found in culture medium or in body fluids, thereby becoming seeds for calcification. The structures described earlier as NB may thus represent remnants and by-products of physiological mechanisms used for calcium homeostasis, a concept which explains the vast body of NB literature as well as explains the true origin of NB as lifeless protein-mineralo entities with questionable role in pathogenesis.     

An ion exchange chromatography and radioimmunoassay procedure for measuring opioid peptides and substance P

The measurements of peptides of the enkephalin, dynorphin and substance P systems is complicated by the number of possible precursor fragments and degradation products that might cross-react with the antisera. By using an ion-exchanger step before radioimmunoassay we can reduce the possibility that observed peptide levels are due to precursors or metabolites. The ion-exchanger method runs with good recovery and its main advantage is that many samples can be run in parallel. The recovery from the ion-exchanger was similar using two different homogenizing media, whereas the measured endogenous levels of [Met] and [Leu]enkephalin were 3–4 fold higher with 1M acetic acid than when a 1:1 MeOH/HCl mixture was used for tissue extraction.     

Lactobacillus reuteri Maintains a Functional Mucosal Barrier during DSS Treatment Despite Mucus Layer Dysfunction

Treatment with the probiotic bacterium Lactobacillus reuteri has been shown to prevent dextran sodium sulfate (DSS)-induced colitis in rats. This is partly due to reduced P-selectin-dependent leukocyte- and platelet-endothelial cell interactions, however, the mechanism behind this protective effect is still unknown. In the present study a combination of culture dependent and molecular based T-RFLP profiling was used to investigate the influence of L. reuteri on the colonic mucosal barrier of DSS treated rats. It was first demonstrated that the two colonic mucus layers of control animals had different bacterial community composition and that fewer bacteria resided in the firmly adherent layer. During DSS induced colitis, the number of bacteria in the inner firmly adherent mucus layer increased and bacterial composition of the two layers no longer differed. In addition, induction of colitis dramatically altered the microbial composition in both firmly and loosely adherent mucus layers. Despite protecting against colitis, treatment with L. reuteri did not improve the integrity of the mucus layer or prevent distortion of the mucus microbiota caused by DSS. However, L. reuteri decreased the bacterial translocation from the intestine to mesenteric lymph nodes during DSS treatment, which might be an important part of the mechanisms by which L. reuteri ameliorates DSS induced colitis.     

Metabolic Effectors Secreted by Bacterial Pathogens: Essential Facilitators of Plastid Endosymbiosis?

Under the endosymbiont hypothesis, over a billion years ago a heterotrophic eukaryote entered into a symbiotic relationship with a cyanobacterium (the cyanobiont). This partnership culminated in the plastid that has spread to forms as diverse as plants and diatoms. However, why primary plastid acquisition has not been repeated multiple times remains unclear. Here, we report a possible answer to this question by showing that primary plastid endosymbiosis was likely to have been primed by the secretion in the host cytosol of effector proteins from intracellular Chlamydiales pathogens. We provide evidence suggesting that the cyanobiont might have rescued its afflicted host by feeding photosynthetic carbon into a chlamydia-controlled assimilation pathway.     

Effect of glycine on the cell yield and growth rate of Escherichia coli: evidence for cell-density-dependent glycine degradation as determined by (13)C NMR spectroscopy.

Addition of selected amino acids could be a means to improve production of recombinant proteins in industrial processes. We found that glycine increased the maximum specific growth rate of Escherichia coli from 0.67 to 0.78 h(-1), and the cell yield from 0.57 to 0.98 g dry weight per g substrate, when supplemented to batch cultures in a glucose-mineral medium. Maximum effect occurred at pH 6.8, at a glycine concentration of 6-12 mmol l(-1), and at cell densities below 1.15 g dry weight l(-1) (0D(610).3). When glycine was added to a culture at a cell density of 1.15 g l(-1) or above, no growth promoting effect of glycine was seen. The 'glycine effect' was not due to CO(2) produced by the glycine cleavage system (GCV), and the lack of effect at higher cell densities was not masked by acetate accumulation, but coincided with increased acetate production. The metabolism of glycine was further investigated in cultures supplied with [2-(13)C] labelled glycine, and the redistribution of label in the [1-(13)C], [2-(13)C], and [1,2-(13)C] isotopomeres of excreted acetate was analysed by 13C NMR. The NMR data revealed that very little degradation of glycine occurred at cell densities below 1.15 g l(-1). Simultaneously the biosynthesis of serine and glycine was repressed as judged by the absence of [2-(13)C] acetate, implying that added glycine was used as a source of glycine, serine, one-carbon units, and threonine. At cell densities above 1.15 g l(-1), 53% of the consumed glycine carbon was excreted as acetate. Degradation of glycine was associated with an increased uptake rate, cleavage by GCV, and degradation of both glycine-derived serine, and glucose-derived serine to pyruvate. This switch in metabolism appears to be regulated by quorum sensing.     

Reduction of inflammation and chronic tissue damage by omega-3 fatty acids in fat-1 transgenic mice with pancreatitis

Pancreatitis is a severe debilitating disease with high morbidity and mortality. Treatment is mostly supportive, and until now there are no clinically useful strategies for anti-inflammatory therapy. Although omega-3 polyunsaturated fatty acids (n-3 PUFA) are known to have anti-inflammatory effects, the utility of these fatty acids in the alleviation of pancreatitis remained to be investigated. The aim of this study was to examine the effect of n-3 PUFA on both acute and chronic pancreatitis in a well-controlled experimental system. We used the fat-1 transgenic mouse model, characterized by endogenously increased tissue levels of n-3 PUFA, and their wild-type littermates to examine the effect of n-3 PUFA on both acute and chronic cerulein-induced pancreatitis. Disease activity and inflammatory status were assessed by both histology and molecular methods. In acute pancreatitis, fat-1 mice showed a trend towards decreased necrosis and significantly reduced levels of plasma IL-6 levels as well as reduced neutrophil infiltration in the lung. In chronic pancreatitis there was less pancreatic fibrosis and collagen content accompanied by decreased pancreatic stellate cell activation in the fat-1 animals with increased n-3 PUFA tissue levels as compared to wild-type littermates with high levels of omega-6 (n-6) PUFA in their tissues. Our data provide evidence for a reduction of systemic inflammation in acute pancreatitis and of tissue fibrosis in chronic pancreatitis by increasing the tissue content of omega-3 polyunsaturated fatty acids. These results suggest a beneficial potential for n-3 PUFA supplementation in acute and particularly chronic pancreatitis.