The retinoblastoma protein regulates pericentric heterochromatin

The retinoblastoma protein (pRb) has been proposed to regulate cell cycle progression in part through its ability to interact with enzymes that modify histone tails and create a repressed chromatin structure. We created a mutation in the murine Rb1 gene that disrupted pRb's ability to interact with these enzymes to determine if it affected cell cycle control. Here, we show that loss of this interaction slows progression through mitosis and causes aneuploidy. Our experiments reveal that while the LXCXE binding site mutation does not disrupt pRb's interaction with the Suv4-20h histone methyltransferases, it dramatically reduces H4-K20 trimethylation in pericentric heterochromatin. Disruption of heterochromatin structure in this chromosomal region leads to centromere fusions, chromosome missegregation, and genomic instability. These results demonstrate the surprising finding that pRb uses the LXCXE binding cleft to control chromatin structure for the regulation of events beyond the G(1)-to-S-phase transition.     

Are large amounts of sodium stored in an osmotically inactive form during sodium retention? Balance studies in freely moving dogs

Alterations in total body sodium (TBSodium) that covered the range from moderate deficit to large surplus were induced by 10 experimental protocols in 66 dogs to study whether large amounts of Na+ are stored in an osmotically inactive form during Na+ retention. Changes in TBSodium, total body potassium (TBPotassium), and total body water (TBWater) were determined by 4-day balance studies. A rather close correlation was found between individual changes in TBSodium and those in TBWater (r2 = 0.83). Changes in TBSodium were often accompanied by changes in TBPotassium. Taking changes of both TBSodium and TBPotassium into account, the correlation with TBWater changes became very close (r2 = 0.93). The sum of changes in TBSodium and TBPotassium was accompanied by osmotically adequate TBWater changes, and plasma osmolality remained unchanged. Calculations reveal that even moderate TBSodium changes often included substantial Na+/K+ exchanges between extracellular and cellular space. The results support the theory that osmocontrol effectively adjusts TBWater to the body's present content of the major cations, Na+ and K+, and do not support the notion that, during Na+ retention, large portions of Na+ are stored in an osmotically inactive form. Furthermore, the finding that TBSodium changes are often accompanied by TBPotassium changes and also include Na+/K+ redistributions between fluid compartments suggests that cells may serve as readily available Na+ store. This Na+ storage, however, is osmotically active, since osmotical equilibration is achieved by opposite redistribution of K+.     

Beyond gastric acid reduction: proton pump inhibitors induce heme oxygenase-1 in gastric and endothelial cells

Proton pump inhibitors (PPIs) have been demonstrated to prevent gastric mucosal injury by mechanisms independent of acid inhibition. Here we demonstrate that both omeprazole and lansoprazole protect human gastric epithelial and endothelial cells against oxidative stress. This effect was abrogated in the presence of the heme oxygenase-1 (HO-1) inhibitor ZnBG. Exposure to either PPI resulted in a strong induction of HO-1 expression on mRNA and protein level, and led to an increased activity of this enzyme. Expression of cyclooxygenase isoforms 1 and 2 remained unaffected, and COX-inhibitors did not antagonize HO-1 induction by PPIs. Our results suggest that the antioxidant defense protein HO-1 is a target of PPIs in both endothelial and gastric epithelial cells. HO-1 induction might account for the gastroprotective effects of PPIs independently of acid inhibition, especially in NSAID gastropathy. Moreover, our findings provide additional perspectives for a possible but yet unexplored use of PPIs in vasoprotection.     

Quantitative analysis of cellular proteome alterations in human influenza A virus‐infected mammalian cell lines

Over the last years virus–host cell interactions were investigated in numerous studies. Viral strategies for evasion of innate immune response, inhibition of cellular protein synthesis and permission of viral RNA and protein production were disclosed. With quantitative proteome technology, comprehensive studies concerning the impact of viruses on the cellular machinery of their host cells at protein level are possible. Therefore, 2‐D DIGE and nanoHPLC‐nanoESI‐MS/MS analysis were used to qualitatively and quantitatively determine the dynamic cellular proteome responses of two mammalian cell lines to human influenza A virus infection. A cell line used for vaccine production (MDCK) was compared with a human lung carcinoma cell line (A549) as a reference model. Analyzing 2‐D gels of the proteomes of uninfected and influenza‐infected host cells, 16 quantitatively altered protein spots (at least ±1.7‐fold change in relative abundance, p<0.001) were identified for both cell lines. Most significant changes were found for keratins, major components of the cytoskeleton system, and for Mx proteins, interferon‐induced key components of the host cell defense. Time series analysis of infection processes allowed the identification of further proteins that are described to be involved in protein synthesis, signal transduction and apoptosis events. Most likely, these proteins are required for supporting functions during influenza viral life cycle or host cell stress response. Quantitative proteome‐wide profiling of virus infection can provide insights into complexity and dynamics of virus–host cell interactions and may accelerate antiviral research and support optimization of vaccine manufacturing processes.     

The peroxisomal receptor Pex19p forms a helical mPTS recognition domain

The protein Pex19p functions as a receptor and chaperone of peroxisomal membrane proteins (PMPs). The crystal structure of the folded C‐terminal part of the receptor reveals a globular domain that displays a bundle of three long helices in an antiparallel arrangement. Complementary functional experiments, using a range of truncated Pex19p constructs, show that the structured α‐helical domain binds PMP‐targeting signal (mPTS) sequences with about 10 μM affinity. Removal of a conserved N‐terminal helical segment from the mPTS recognition domain impairs the ability for mPTS binding, indicating that it forms part of the mPTS‐binding site. Pex19p variants with mutations in the same sequence segment abolish correct cargo import. Our data indicate a divided N‐terminal and C‐terminal structural arrangement in Pex19p, which is reminiscent of a similar division in the Pex5p receptor, to allow separation of cargo‐targeting signal recognition and additional functions.     

A proteomic approach towards the identification of the matrix protein content of the two types of microbodies in Neurospora crassa

Microbodies (peroxisomes) comprise a class of organelles with a similar biogenesis but remarkable biochemical heterogeneity. Here, we purified the two distinct microbody family members of filamentous fungi, glyoxysomes and Woronin bodies, from Neurospora crassa and analyzed their protein content by HPLC/ESI‐MS/MS. In the purified Woronin bodies, we unambiguously identified only hexagonal 1 (HEX1), suggesting that the matrix is probably exclusively filled with the HEX1 hexagonal crystal. The proteomic analysis of highly purified glyoxysomes allowed the identification of 191 proteins. Among them were 16 proteins with a peroxisomal targeting signal type 1 (PTS1) and three with a PTS2. The collection also contained the previously described N. crassa glyoxysomal matrix proteins FOX2 and ICL1 that lack a typical PTS. Three PTS1 proteins were identified that likely represent the long sought glyoxysomal acyl‐CoA dehydrogenases of filamentous fungi. Two of them were demonstrated by subcellular localization studies to be indeed glyoxysomal. Furthermore, two PTS proteins were identified that are suggested to be involved in the detoxification of nitroalkanes. Since the glyoxysomal localization was experimentally demonstrated for one of these enzymes, a new biochemical reaction is expected to be associated with microbody function.     

Sequence-specific 1H, 13C, and 15N assignment of the extended PDZ3 domain of the protein tyrosine phosphatase basophil-like PTP-BL

Protein tyrosine phosphatase basophil-like (PTP-BL), also known as PTPN13, represents a large multi domain non-transmembrane scaffolding protein that contains five PDZ domains. Here we report the complete resonance assignments of the extended PDZ3 domain of PTP-BL. These assignments provide a basis for the detailed structural investigation of the interaction between the PDZ domains of PTP-BL as well as of their interaction with ligands. It will also lead to a better understanding of the proposed scaffolding function of these domains in multi-protein complexes assembled by PTB-BL.     

Influence of roughage/concentrate ratio and linseed oil on the concentration of trans-fatty acids and conjugated linoleic acid in duodenal chyme and milk fat of late lactating cows

The objective of the study was to investigate the influence of two roughage-to-concentrate ratios, with or without linseed oil supplementation, on the flow of fatty acids in the intestinal chyme and the secretion in milk fat in late lactating cows. Seven late lactating cows fitted with cannulae in the dorsal rumen and simple T-shaped cannulae in the proximal duodenum were randomly assigned to four experimental periods applying an incomplete replicated 2 x 2 Latin square design. The rations consisted of meadow hay and a concentrate mixture given in a ratio of 70:30 or 30:70 on dry matter basis. The basal rations were fed without or with 200 g linseed oil daily. After three weeks of adaptation, samples from the duodenal chyme were taken to study the flow of fatty acids. Additionally, milk samples were analysed for their milk fat composition. Decreasing roughage/concentrate ratio and linseed oil supplementation significantly increased the flow of monounsaturated fatty acids (MUFA), trans-fatty acids (tFA) and conjugated linoleic acids (CLA) in the duodenum. Furthermore, linseed oil increased the flow of saturated fatty acids (SFA) in the duodenum. Higher concentrate portion (H 30) and linseed oil supplementation significantly decreased the milk fat content. SFA were lower (p < 0.05) and MUFA were higher (p < 0.05) in milk fat after linseed oil supplementation; H 30 resulted in more polyunsaturated fatty acids (PUFA, p < 0.05) in the milk. Linseed oil supplementation significantly increased tFA and CLA in milk fat. The higher CLA content in milk fat as compared to that in the digesta suggests that a substantial endogenous synthesis of CLA in the mammary gland tissue through A9-desaturase took place. Between 21% and 48% of duodenal t11-C(18:1) were converted into c9, t11-CLA in milk fat.     

Substitution in position 3 of cyclosporin A abolishes the cyclophilin-mediated gain-of-function mechanism but not immunosuppression

Binary complex formation between the immunosuppressive drug cyclosporin A (CsA) and cyclophilin 18 is the prerequisite for the ability of CsA to inhibit the protein phosphatase activity of calcineurin, a central mediator of antigen-receptor signaling. We show here that several CsA derivatives substituted in position 3 can inhibit calcineurin without prior formation of a complex with cyclophilin 18. [Methylsarcosine(3)]CsA was shown to inhibit calcineurin, either in its free form with an IC(50) value of 10 microm, or in its complex form with cyclophilin 18 with an IC(50) of 500 nm. [Dimethylaminoethylthiosarcosine(3)]CsA ([Dat-Sar(3)]CsA) was found to inhibit calcineurin on its own, with an IC(50) value of 1.0 microm, but was not able to inhibit calcineurin after forming the [Dat-Sar(3)]CsA-cyclophilin 18 binary complex. Despite their different inhibitory properties, both CsA and [Dat-Sar(3)]CsA suppressed T cell proliferation and cytokine production mainly through blocking NFAT activation and interleukin-2 gene expression. Furthermore, to demonstrate that [Dat-Sar(3)]CsA can inhibit calcineurin in a cyclophilin-independent manner in vivo, we tested its effect in a Saccharomyces cerevisiae strain (Delta12), in which all the 12 cyclophilins and FKBPs were deleted. [Dat-Sar(3)]CsA, but not CsA, bypassed the requirement for cellular cyclophilins and caused growth inhibition in the salt-stressed Delta12 strain.