Gas vesicles isolated from Halobacterium cells by lysis in hypotonic solution are structurally weakened

Analysis of pressure-collapse curves of Halobacterium cells containing gas vesicles and of gas vesicles released from such cells by hypotonic lysis shows that the isolated gas vesicles are considerably weaker than those present within the cells: their mean critical collapse pressure was around 0.049-0.058 MPa, as compared to 0.082-0.095 MPa for intact cells. The hypotonic lysis procedure, which is widely used for the isolation of gas vesicles from members of the Halobacteriaceae, thus damages the mechanical properties of the vesicles. The phenomenon can possibly be attributed to the loss of one or more structural gas vesicle proteins such as GvpC, the protein that strengthens the vesicles built of GvpA subunits: Halobacterium GvpC is a highly acidic, typically "halophilic" protein, expected to denature in the absence of molar concentrations of salt.     

PLA2G6 mutation underlies infantile neuroaxonal dystrophy

Infantile neuroaxonal dystrophy (INAD) is an autosomal recessive progressive neurodegenerative disease that presents within the first 2 years of life and culminates in death by age 10 years. Affected individuals from two unrelated Bedouin Israeli kindreds were studied. Brain imaging demonstrated diffuse cerebellar atrophy and abnormal iron deposition in the medial and lateral globus pallidum. Progressive white-matter disease and reduction of the N-acetyl aspartate : chromium ratio were evident on magnetic resonance spectroscopy, suggesting loss of myelination. The clinical and radiological diagnosis of INAD was verified by sural nerve biopsy. The disease gene was mapped to a 1.17-Mb locus on chromosome 22q13.1 (LOD score 4.7 at recombination fraction 0 for SNP rs139897), and an underlying mutation common to both affected families was identified in PLA2G6, the gene encoding phospholipase A2 group VI (cytosolic, calcium-independent). These findings highlight a role of phospholipase in neurodegenerative disorders.     

Buoyancy studies in natural communities of square gas-vacuolate archaea in saltern crystallizer ponds

Background

Possession of gas vesicles is generally considered to be advantageous to halophilic archaea: the vesicles are assumed to enable the cells to float, and thus reach high oxygen concentrations at the surface of the brine.

Results

We studied the possible ecological advantage of gas vesicles in a dense community of flat square extremely halophilic archaea in the saltern crystallizer ponds of Eilat, Israel. We found that in this environment, the cells' content of gas vesicles was insufficient to provide positive buoyancy. Instead, sinking/floating velocities were too low to permit vertical redistribution.

Conclusion

The hypothesis that the gas vesicles enable the square archaea to float to the surface of the brines in which they live was not supported by experimental evidence. Presence of the vesicles, which are mainly located close to the cell periphery, may provide an advantage as they may aid the cells to position themselves parallel to the surface, thereby increasing the efficiency of light harvesting by the retinal pigments in the membrane.     

A Dominant Negative Heterozygous G87R Mutation in the Zinc Transporter, ZnT-2 (SLC30A2), Results in Transient Neonatal Zinc Deficiency

Zinc is an essential mineral, and infants are particularly vulnerable to zinc deficiency as they require large amounts of zinc for their normal growth and development. We have recently described the first loss-of-function mutation (H54R) in the zinc transporter ZnT-2 (SLC30A2) in mothers with infants harboring transient neonatal zinc deficiency (TNZD). Here we identified and characterized a novel heterozygous G87R ZnT-2 mutation in two unrelated Ashkenazi Jewish mothers with infants displaying TNZD. Transient transfection of G87R ZnT-2 resulted in endoplasmic reticulum-Golgi retention, whereas the WT transporter properly localized to intracellular secretory vesicles in HC11 and MCF-7 cells. Consequently, G87R ZnT-2 showed decreased stability compared with WT ZnT-2 as revealed by Western blot analysis. Three-dimensional homology modeling based on the crystal structure of YiiP, a close zinc transporter homologue from Escherichia coli, revealed that the basic arginine residue of the mutant G87R points toward the membrane lipid core, suggesting misfolding and possible loss-of-function. Indeed, functional assays including vesicular zinc accumulation, zinc secretion, and cytoplasmic zinc pool assessment revealed markedly impaired zinc transport in G87R ZnT-2 transfectants. Moreover, co-transfection experiments with both mutant and WT transporters revealed a dominant negative effect of G87R ZnT-2 over the WT ZnT-2; this was associated with mislocalization, decreased stability, and loss of zinc transport activity of the WT ZnT-2 due to homodimerization observed upon immunoprecipitation experiments. These findings establish that inactivating ZnT-2 mutations are an underlying basis of TNZD and provide the first evidence for the dominant inheritance of heterozygous ZnT-2 mutations via negative dominance due to homodimer formation.     

Semaphorin 3A is a marker for disease activity and a potential immunoregulator in systemic lupus erythematosus

Introduction

Semaphorin 3A (sema3A) and neuropilin-1 (NP-1) play a regulatory role in immune responses and have a demonstrated effect on the course of collagen induced arthritis. This study was undertaken to evaluate the role of sema3A and NP-1 in the pathogenesis of systemic lupus erythematosus (SLE) and the specific effect of sema3A on the auto-reactive properties of B cells in SLE patients.

Methods

Thirty two SLE and 24 rheumatoid arthritis (RA) patients were assessed and compared with 40 normal individuals. Sema3A serum levels were measured and correlated with SLE disease activity. The in vitro effect of sema3A in reducing Toll-like receptor 9 (TLR-9) expression in B cells of SLE patients was evaluated.

Results

Sema3A serum levels in SLE patients were found to be significantly lower than in RA patients (55.04 ± 16.30 ng/ml versus 65.54 ± 14.82 ng/ml, P = 0.018) and lower yet than in normal individuals (55.04 ± 16.30 ng/ml versus 74.41 ± 17.60 ng/ml, P < 0.0001). Altered serum sema3A levels were found to be in inverse correlation with SLE disease activity, mainly with renal damage. The expression of both sema3A and NP-1 on B cells from SLE patients was significantly different in comparison with normal healthy individuals. Finally, when sema3A was co-cultured with cytosine-phosphodiester-guanine oligodeoxynucleotides (CpG-ODN)-stimulated B cells of SLE patients, their TLR-9 expression was significantly reduced, by almost 50% (P = 0.001).

Conclusions

This is the first study in which a reduced serum level of sema3A was found in association with SLE disease activity. It also raises the possibility that sema3A may have a regulatory function in SLE.     

Microbial and chemical characterization of underwater fresh water springs in the Dead Sea

Due to its extreme salinity and high Mg concentration the Dead Sea is characterized by a very low density of cells most of which are Archaea. We discovered several underwater fresh to brackish water springs in the Dead Sea harboring dense microbial communities. We provide the first characterization of these communities, discuss their possible origin, hydrochemical environment, energetic resources and the putative biogeochemical pathways they are mediating. Pyrosequencing of the 16S rRNA gene and community fingerprinting methods showed that the spring community originates from the Dead Sea sediments and not from the aquifer. Furthermore, it suggested that there is a dense Archaeal community in the shoreline pore water of the lake. Sequences of bacterial sulfate reducers, nitrifiers iron oxidizers and iron reducers were identified as well. Analysis of white and green biofilms suggested that sulfide oxidation through chemolitotrophy and phototrophy is highly significant. Hyperspectral analysis showed a tight association between abundant green sulfur bacteria and cyanobacteria in the green biofilms. Together, our findings show that the Dead Sea floor harbors diverse microbial communities, part of which is not known from other hypersaline environments. Analysis of the water's chemistry shows evidence of microbial activity along the path and suggests that the springs supply nitrogen, phosphorus and organic matter to the microbial communities in the Dead Sea. The underwater springs are a newly recognized water source for the Dead Sea. Their input of microorganisms and nutrients needs to be considered in the assessment of possible impact of dilution events of the lake surface waters, such as those that will occur in the future due to the intended establishment of the Red Sea-Dead Sea water conduit.     

Microbial life at high salt concentrations: phylogenetic and metabolic diversity

Halophiles are found in all three domains of life. Within the Bacteria we know halophiles within the phyla Cyanobacteria, Proteobacteria, Firmicutes, Actinobacteria, Spirochaetes, and Bacteroidetes. Within the Archaea the most salt-requiring microorganisms are found in the class Halobacteria. Halobacterium and most of its relatives require over 100–150 g/l salt for growth and structural stability. Also within the order Methanococci we encounter halophilic species. Halophiles and non-halophilic relatives are often found together in the phylogenetic tree, and many genera, families and orders have representatives with greatly different salt requirement and tolerance. A few phylogenetically coherent groups consist of halophiles only: the order Halobacteriales, family Halobacteriaceae (Euryarchaeota) and the anaerobic fermentative bacteria of the order Halanaerobiales (Firmicutes). The family Halomonadaceae (Gammaproteobacteria) almost exclusively contains halophiles. Halophilic microorganisms use two strategies to balance their cytoplasm osmotically with their medium. The first involves accumulation of molar concentrations of KCl. This strategy requires adaptation of the intracellular enzymatic machinery, as proteins should maintain their proper conformation and activity at near-saturating salt concentrations. The proteome of such organisms is highly acidic, and most proteins denature when suspended in low salt. Such microorganisms generally cannot survive in low salt media. The second strategy is to exclude salt from the cytoplasm and to synthesize and/or accumulate organic 'compatible' solutes that do not interfere with enzymatic activity. Few adaptations of the cells' proteome are needed, and organisms using the 'organic-solutes-in strategy' often adapt to a surprisingly broad salt concentration range. Most halophilic Bacteria, but also the halophilic methanogenic Archaea use such organic solutes. A variety of such solutes are known, including glycine betaine, ectoine and other amino acid derivatives, sugars and sugar alcohols. The 'high-salt-in strategy' is not limited to the Halobacteriaceae. The Halanaerobiales (Firmicutes) also accumulate salt rather than organic solutes. A third, phylogenetically unrelated organism accumulates KCl: the red extremely halophilic Salinibacter (Bacteroidetes), recently isolated from saltern crystallizer brines. Analysis of its genome showed many points of resemblance with the Halobacteriaceae, probably resulting from extensive horizontal gene transfer. The case of Salinibacter shows that more unusual types of halophiles may be waiting to be discovered.     

Upregulation of the let-7 microRNA with precocious development in lin-12/Notch hypermorphic Caenorhabditis elegans mutants

The lin-12/Notch signaling pathway is conserved from worms to humans and is a master regulator of metazoan development. Here, we demonstrate that lin-12/Notch gain-of-function (gf) animals display precocious alae at the L4 larval stage with a significant increase in let-7 expression levels. Furthermore, lin-12(gf) animals display a precocious and higher level of let-7 gfp transgene expression in seam cells at L3 stage. Interestingly, lin-12(gf) mutant rescued the lethal phenotype of let-7 mutants similar to other known heterochronic mutants. We propose that lin-12/Notch signaling pathway functions in late developmental timing, upstream of or in parallel to the let-7 heterochronic pathway. Importantly, the human microRNA let-7a was also upregulated in various human cell lines in response to Notch1 activation, suggesting an evolutionarily conserved cross-talk between let-7 and the canonical lin-12/Notch signaling pathway.