Autoantibodies directed to novel components of the PM/Scl complex, the human exosome

The autoantigenic polymyositis/scleroderma (PM/Scl) complex was recently shown to be the human homologue of the yeast exosome, which is an RNA-processing complex. Our aim was to assess whether, in addition to targeting the known autoantigens PM/Scl-100 and PM/Scl-75, autoantibodies also target recently identified components of the PM/Scl complex. The prevalence of autoantibodies directed to six novel human exosome components (hRrp4p, hRrp40p, hRrp41p, hRrp42p, hRrp46p, hCsl4p) was determined in sera from patients with idiopathic inflammatory myopathy (n = 48), scleroderma (n = 11), or the PM/Scl overlap syndrome (n = 10). The sera were analyzed by enzyme-linked immunosorbent assays and western blotting using the affinity-purified recombinant proteins. Our results show that each human exosome component is recognized by autoantibodies. The hRrp4p and hRrp42p components were most frequently targeted. The presence of autoantibodies directed to the novel components of the human exosome was correlated with the presence of the anti-PM/Scl-100 autoantibody in the sera of patients with idiopathic inflammatory myopathy (IIM), as was previously found for the anti-PM/Scl-75 autoantibody. Other clear associations between autoantibody activities were not found. These results further support the conception that the autoimmune response may initially be directed to PM/Scl-100, whereas intermolecular epitope spreading may have caused the autoantibody response directed to the associated components.     

Lactate dehydrogenase A as a highly abundant eye lens protein in platypus (Ornithorhynchus anatinus): upsilon (upsilon)-crystallin

Vertebrate eye lenses mostly contain two abundant types of proteins, the alpha-crystallins and the beta/gamma-crystallins. In addition, certain housekeeping enzymes are highly expressed as crystallins in various taxa. We now observed an unusual approximately 41-kd protein that makes up 16% to 18% of the total protein in the platypus eye lens. Its cDNA sequence was determined, which identified the protein as muscle-type lactate dehydrogenase A (LDH-A). It is the first observation of LDH-A as a crystallin, and we designate it upsilon (upsilon)-crystallin. Interestingly, the related heart-type LDH-B occurs as an abundant lens protein, known as epsilon-crystallin, in many birds and crocodiles. Thus, two members of the ldh gene family have independently been recruited as crystallins in different higher vertebrate lineages, suggesting that they are particularly suited for this purpose in terms of gene regulatory or protein structural properties. To establish whether platypus LDH-A/upsilon-crystallin has been under different selective constraints as compared with other vertebrate LDH-A sequences, we reconstructed the vertebrate ldh-a gene phylogeny. No conspicuous rate deviations or amino acid replacements were observed.     

Caspartin and calprismin, two proteins of the shell calcitic prisms of the Mediterranean fan mussel Pinna nobilis

We used the combination of preparative electrophoresis and immunological detection to isolate two new proteins from the shell calcitic prisms of Pinna nobilis, the Mediterranean fan mussel. The amino acid composition of these proteins was determined. Both proteins are soluble, intracrystalline, and acidic. The 38-kDa protein is glycosylated; the 17-kDa one is not. Ala, Asx, Thr, and Pro represent the dominant residues of the 38-kDa protein, named calprismin. An N-terminal sequence was obtained from calprismin. This sequence, which comprises a pattern of 4 cysteine residues, is not related to any known protein. The second protein, named caspartin, exhibits an unusual amino acid composition, since Asx constitutes by far the main amino acid residue. Preliminary sequencing surprisingly suggests that the first 75 N-terminal residues are all Asp. Caspartin self-aggregates spontaneously into multimers. In vitro tests show that it inhibits the precipitation of calcium carbonate. Furthermore, it strongly interferes with the growth of calcite crystals. A polyclonal antiserum raised against caspartin was used to localize this protein in the shell by immunogold. The immunolocalization demonstrates that caspartin is distributed within the prisms and makes a continuous film at the interface between the prisms and the surrounding insoluble sheets. Our finding emphasizes the prominent role of aspartic acid-rich proteins for the building of calcitic prisms among molluscs.     

Genome-Wide Expression Profiling Identifies Type 1 Interferon Response Pathways in Active Tuberculosis

Tuberculosis (TB), caused by Mycobacterium tuberculosis (M.tb), remains the leading cause of mortality from a single infectious agent. Each year around 9 million individuals newly develop active TB disease, and over 2 billion individuals are latently infected with M.tb worldwide, thus being at risk of developing TB reactivation disease later in life. The underlying mechanisms and pathways of protection against TB in humans, as well as the dynamics of the host response to M.tb infection, are incompletely understood. We carried out whole-genome expression profiling on a cohort of TB patients longitudinally sampled along 3 time-points: during active infection, during treatment, and after completion of curative treatment. We identified molecular signatures involving the upregulation of type-1 interferon (α/β) mediated signaling and chronic inflammation during active TB disease in an Indonesian population, in line with results from two recent studies in ethnically and epidemiologically different populations in Europe and South Africa. Expression profiles were captured in neutrophil-depleted blood samples, indicating a major contribution of lymphocytes and myeloid cells. Expression of type-1 interferon (α/β) genes mediated was also upregulated in the lungs of M.tb infected mice and in infected human macrophages. In patients, the regulated gene expression-signature normalized during treatment, including the type-1 interferon mediated signaling and a concurrent opposite regulation of interferon-gamma. Further analysis revealed IL15RA, UBE2L6 and GBP4 as molecules involved in the type-I interferon response in all three experimental models. Our data is highly suggestive that the innate immune type-I interferon signaling cascade could be used as a quantitative tool for monitoring active TB disease, and provide evidence that components of the patient’s blood gene expression signature bear similarities to the pulmonary and macrophage response to mycobacterial infection.     

Hemizygous deletion of CTGF/CCN2 does not suffice to prevent fibrosis of the severely injured kidney

BackgroundConnective Tissue Growth Factor (CTGF/CCN2) is an important mediator of kidney fibrosis. Previous observations indicated that attenuation of CCN2 expression sufficed to alleviate early kidney damage. However, little is known about the role of CCN2 in fibrosis of severely damaged and more chronically injured kidneys. Therefore, we examined the effects of CCN2 haploinsufficiency on the progression of renal scarring in long-term STZ-induced diabetic nephropathy, in a more advanced stage of obstructive nephropathy following unilateral ureteric obstruction (UUO), and in severe aristolochic acid (AA)-induced tubulotoxic nephritis.MethodsWild-type (WT, CCN2^+/+) and hemizygous CCN2^+/? C57Bl/6 mice were studied. In the diabetes experiment, streptozotocin-injected and control mice were followed for 6 months, with regular blood pressure, glycaemia and albuminuria recordings. In the UUO experiment, the left ureter was obstructed for 14 days with the contralateral kidney serving as control. For the AA experiment, mice were followed for 25 days after 5 intraperitoneal injections with AA and compared to control mice injected with buffer alone. Organs were harvested for histology, mRNA and protein measurements. Collagen content was determined by HPLC and expressed as hydroxyproline/proline ratio.ResultsCCN2 expression was significantly increased in the damaged as compared to control kidneys. In all three models, CCN2 levels in the damaged kidneys of CCN2^+/? mice averaged about 50% of those in damaged WT kidneys. After 6 months of diabetes, albuminuria was increased 2.5-fold in WT mice, compared to 1.5-fold in CCN2^+/? mice, mesangial matrix was expanded 5-fold in WT and 4.4-fold in CCN2^+/? mice and the glomerular basement membrane was thickened 1.3-fold in WT and 1.5-fold in CCN2^+/? mice (all differences between WT and CCN2^+/? mice are NS). Tubular damage and interstitial fibrosis scores were also not different between Wt and CCN2^+/? mice in the diabetes (1.8 vs. 1.7), UUO (2.8 vs. 2.6), and AA (1.4 vs. 1.2) models, as was the case for macrophage influx and collagen content in these three models.ConclusionUnlike in mild and relatively early STZ-induced diabetic nephropathy, scarring of severely and chronically damaged kidneys is not attenuated by a 50% reduction of CCN2 to (near) normal levels. This suggests that CCN2 is either redundant in severe and chronic kidney disease, or that it is a limiting factor only at subnormal concentrations requiring further reduction by available or emerging therapies to prevent fibrosis of the severely injured kidney.     

Maximizing affluence within the planetary boundaries

Purpose

Ordinary product LCA studies focus on measuring or minimizing environmental impact, but do not address if the product fits in a sustainable consumption pattern. This paper proposes a setup in which the planetary boundaries define the maximum impact, and the minimum requirements for a reasonable consumption level specify a lower impact level. Thus, a “safe operating space” remains.

Methods

We use an IO table for EU-27 and the consumption pattern of the Bulgarian population extrapolated to the EU level as driving climate impact. The EU’s policy targets are used as a planetary boundary for climate change.

Results

The 2020 target is shown to be able to accommodate the Bulgarian-style consumption, with room for a much higher GDP. The 2050 target, however, is too narrow, and a slightly smaller consumption pattern is needed to reach the target.

Conclusions

Although the approach is highly simplified and neglects many developments, the idea of using IO-tables and minimum consumption levels to backcast directions to be taken is expected to help policymakers. We acknowledge some important limitations of our approach, but accept these in the context of exploring future scenarios and how to get there, instead of predicting the future.     

The human peptidylarginine deiminases type 2 and type 4 have distinct substrate specificities

Human peptidylarginine deiminases (hPADs) have been implicated in several diseases, particularly in rheumatoid arthritis. Since hPAD2 and hPAD4 are the isotypes expressed in the inflamed joints of RA patients and protein citrullination by PADs has been proposed to play a pathophysiological role, they represent unique therapeutic targets. To facilitate the development of substrate-based PAD inhibitors the substrate specificity of hPAD2 and hPAD4 was determined. Recombinant hPADs were expressed in bacteria or mammalian cell lines and allowed to citrullinate proteins in cell lysates, as well as a series of synthetic peptides. The citrullinated residues in proteins and the efficiency of peptide citrullination were determined by mass spectrometry. In total 320 hPAD2 and 178 hPAD4 citrullination sites were characterized. Amino acid residues most commonly found in citrullination sites for both isotypes are Gly at + 1 and Tyr at + 3 relative to the target arginine. For hPAD4 several additional amino acids were observed to be preferred at various positions from − 4 to + 4. The substrate motifs determined by amino acid substitution analysis partially confirmed these preferences, although peptide context dependent differences were also observed. Taken together, our data show that the enzyme specificity for cellular substrates and synthetic peptides differs for hPAD2 and hPAD4. hPAD4 shows more restrictive substrate specificity compared to hPAD2. Consensus sequences, which can be used as the basis for the development of PAD inhibitors, were derived for the citrullination sites of both hPAD2 and hPAD4.     

Sorbitol production from lactose by engineered Lactobacillus casei deficient in sorbitol transport system and mannitol-1-phosphate dehydrogenase

Sorbitol is a sugar alcohol largely used in the food industry as a low-calorie sweetener. We have previously described a sorbitol-producing Lactobacillus casei (strain BL232) in which the gutF gene, encoding a sorbitol-6-phosphate dehydrogenase, was expressed from the lactose operon. Here, a complete deletion of the ldh1 gene, encoding the main l-lactate dehydrogenase, was performed in strain BL232. In a resting cell system with glucose, the new strain, named BL251, accumulated sorbitol in the medium that was rapidly metabolized after glucose exhaustion. Reutilization of produced sorbitol was prevented by deleting the gutB gene of the phosphoenolpyruvate: sorbitol phosphotransferase system (PTS^Gut) in BL251. These results showed that the PTS^Gut did not mediate sorbitol excretion from the cells, but it was responsible for uptake and reutilization of the synthesized sorbitol. A further improvement in sorbitol production was achieved by inactivation of the mtlD gene, encoding a mannitol-1-phosphate dehydrogenase. The new strain BL300 (lac::gutF Δldh1 ΔgutB mtlD) showed an increase in sorbitol production whereas no mannitol synthesis was detected, avoiding thus a polyol mixture. This strain was able to convert lactose, the main sugar from milk, into sorbitol, either using a resting cell system or in growing cells under pH control. A conversion rate of 9.4% of lactose into sorbitol was obtained using an optimized fed-batch system and whey permeate, a waste product of the dairy industry, as substrate.     

Synthesis and characterization of an unusual equatorially substituted di-manganese compound. The first structural determination of a phosphite compound of type [Mn2(CO)9P]

Reaction of (acetonitrile)-nona(carbonyl)-di-manganese and tri-iso-propyl phosphite provided the new phosphite compound [Mn₂(CO)₉L]. The product was fully characterized and the molecular structure was determined by single crystal X-ray diffraction. This represents the first structural analysis of a di-manganese carbonyl compound containing phosphite ligands. In addition, this compound displays a rare example of an equatorially substituted di-manganese carbonyl compound.