Ltv1 is required for efficient nuclear export of the ribosomal small subunit in Saccharomyces cerevisiae

In eukaryotes, 40S and 60S ribosomal subunits are assembled in the nucleus and exported to the cytoplasm independently of one another. Nuclear export of the 60S requires the adapter protein Nmd3, but no analogous adapter has been identified for the 40S. Ltv1 is a nonessential, nonribosomal protein that is required for 40S subunit biogenesis in yeast. Cells lacking LTV1 grow slowly, are hypersensitive to inhibitors of protein synthesis, and produce about half as many 40S subunits as do wild-type cells. Ltv1 interacts with Crm1, co-sediments in sucrose gradients with 43S/40S subunits, and copurifies with late 43S particles. Here we show that Ltv1 shuttles between nucleus and cytoplasm in a Crm1-dependent manner and that it contains a functional NES that is sufficient to direct the export of an NLS-containing reporter. Small subunit export is reduced in Deltaltv1 mutants, as judged by the altered distribution of the 5'-ITS1 rRNA and the 40S ribosomal protein RpS3. Finally, we show a genetic interaction between LTV1 and YRB2, a gene that encodes a Ran-GTP-, Crm1-binding protein that facilitates the small subunit export. We propose that Ltv1 functions as one of several possible adapter proteins that link the nuclear export machinery to the small subunit.     

The F-box protein Dia2 overcomes replication impedance to promote genome stability in Saccharomyces cerevisiae

The maintenance of DNA replication fork stability under conditions of DNA damage and at natural replication pause sites is essential for genome stability. Here, we describe a novel role for the F-box protein Dia2 in promoting genome stability in the budding yeast Saccharomyces cerevisiae. Like most other F-box proteins, Dia2 forms a Skp1-Cdc53/Cullin-F-box (SCF) E3 ubiquitin–ligase complex. Systematic analysis of genetic interactions between dia2Δ and ~4400 viable gene deletion mutants revealed synthetic lethal/synthetic sick interactions with a broad spectrum of DNA replication, recombination, checkpoint, and chromatin-remodeling pathways. dia2Δ strains exhibit constitutive activation of the checkpoint kinase Rad53 and elevated counts of endogenous DNA repair foci and are unable to overcome MMS-induced replicative stress. Notably, dia2Δ strains display a high rate of gross chromosomal rearrangements (GCRs) that involve the rDNA locus and an increase in extrachromosomal rDNA circle (ERC) formation, consistent with an observed enrichment of Dia2 in the nucleolus. These results suggest that Dia2 is essential for stable passage of replication forks through regions of damaged DNA and natural fragile regions, particularly the replication fork barrier (RFB) of rDNA repeat loci. We propose that the SCF^Dia2 ubiquitin ligase serves to modify or degrade protein substrates that would otherwise impede the replication fork in problematic regions of the genome.     

Functional hierarchy of the N-terminal tyrosines of SLP-76

The adaptor protein Src homology 2 domain-containing leukocyte phosphoprotein of 76 kDa (SLP-76) plays a central role in T cell activation and T cell development. SLP-76 has three functional modules: an acidic domain with three key tyrosines, a central proline-rich domain, and a C-terminal Src homology 2 domain. Of these, mutation of the three N-terminal tyrosines (Y112, Y128, and Y145) results in the most profound effects on T cell development and function. Y112 and Y128 associate with Vav and Nck, two proteins shown to be important for TCR-induced phosphorylation of proximal signaling substrates, Ca(2+) flux, and actin reorganization. Y145 has been shown to be important for optimal association of SLP-76 with inducible tyrosine kinase, a key regulator of T cell function. To investigate further the role of the phosphorylatable tyrosines of SLP-76 in TCR signaling, cell lines and primary T cells expressing SLP-76 with mutations in individual or paired tyrosine residues were analyzed. These studies show that Tyr(145) of SLP-76 is the most critical tyrosine for both T cell function in vitro and T cell development in vivo.     

Macrophage migration inhibitory factor induces macrophage recruitment via CC chemokine ligand 2

Macrophage migration inhibitory factor (MIF) was originally identified for its ability to inhibit the random migration of macrophages in vitro. MIF is now recognized as an important mediator in a range of inflammatory disorders. We recently observed that the absence of MIF is associated with a reduction in leukocyte-endothelial cell interactions induced by a range of inflammatory mediators, suggesting that one mechanism whereby MIF acts during inflammatory responses is by promoting leukocyte recruitment. However, it is unknown whether MIF is capable of inducing leukocyte recruitment independently of additional inflammatory stimuli. In this study, we report that MIF is capable of inducing leukocyte adhesion and transmigration in postcapillary venules in vivo. Moreover, leukocytes recruited in response to MIF were predominantly CD68(+) cells of the monocyte/macrophage lineage. Abs against the monocyte-selective chemokine CCL2 (JE/MCP-1) and its receptor CCR2, but not CCL3 and CXCL2, significantly inhibited MIF-induced monocyte adhesion and transmigration. CCL2(-/-) mice displayed a similar reduction in MIF-induced recruitment indicating a critical role of CCL2 in the MIF-induced response. This hypothesis was supported by findings that MIF induced CCL2 release from primary microvascular endothelial cells. These data demonstrate a previously unrecognized function of this pleiotropic cytokine: induction of monocyte migration into tissues. This function may be critical to the ability of MIF to promote diseases such as atherosclerosis and rheumatoid arthritis, in which macrophages are key participants.     

Oxidative modification of M-type K(+) channels as a mechanism of cytoprotective neuronal silencing

Voltage-gated K(+) channels of the Kv7 family underlie the neuronal M current that regulates action potential firing. Suppression of M current increases excitability and its enhancement can silence neurons. We here show that three of five Kv7 channels undergo strong enhancement of their activity by oxidative modification induced by physiological concentrations of hydrogen peroxide. A triple cysteine pocket in the channel S2-S3 linker is critical for this effect. Oxidation-induced enhancement of M current produced a hyperpolarization and a dramatic reduction of action potential firing frequency in rat sympathetic neurons. As hydrogen peroxide is robustly produced during hypoxia-induced oxidative stress, we used an oxygen/glucose deprivation neurodegeneration model that showed neuronal death to be severely accelerated by M current blockade. Such blockade had no effect on survival of normoxic neurons. This work describes a novel pathway of M-channel regulation and suggests a role for M channels in protective neuronal silencing during oxidative stress.     

Generation and characterization of B7-H4/B7S1/B7x-deficient mice

Members of the B7 family of cosignaling molecules regulate T-cell proliferation and effector functions by engaging cognate receptors on T cells. In vitro and in vivo blockade experiments indicated that B7-H4 (also known as B7S1 or B7x) inhibits proliferation, cytokine production, and cytotoxicity of T cells. B7-H4 binds to an unknown receptor(s) that is expressed on activated T cells. However, whether B7-H4 plays nonredundant immune regulatory roles in vivo has not been tested. We generated B7-H4-deficient mice to investigate the roles of B7-H4 during various immune reactions. Consistent with its inhibitory function in vitro, B7-H4-deficient mice mounted mildly augmented T-helper 1 (Th1) responses and displayed slightly lowered parasite burdens upon Leishmania major infection compared to the wild-type mice. However, the lack of B7-H4 did not affect hypersensitive inflammatory responses in the airway or skin that are induced by either Th1 or Th2 cells. Likewise, B7-H4-deficient mice developed normal cytotoxic T-lymphocyte reactions against viral infection. Thus, B7-H4 plays a negative regulatory role in vivo but the impact of B7-H4 deficiency is minimal. These results suggest that B7-H4 is one of multiple negative cosignaling molecules that collectively provide a fine-tuning mechanism for T-cell-mediated immune responses.     

Determination of adenine and pyridine nucleotides in glucose-limited chemostat cultures of Penicillium simplicissimum by one-step ethanol extraction and ion-pairing liquid chromatography

Under specific conditions Penicillium simplicissimum excretes large amounts of organic acids, mainly citrate. As the energetic status of the hyphae might play a role in that respect, we developed a method for the determination of adenine (adenosine triphosphate, adenosine diphosphate, and adenosine monophosphate) and pyridine (nicotinamide adenine dinucleotide and reduced nicotinamide adenine dinucleotide (NADH)) nucleotides in hyphae of P. simplicissimum. An optimum separation of the five compounds in less than 15 min was possible on a C-8 column, utilizing 50 mM aqueous triethylamine-buffer (pH 6.5) and acetonitrile as mobile phase; detection was performed at 254 nm. With the exception of NADH, which could not be determined accurately due to stability problems, the method was sensitive (LOD < or = 0.7 ng on-column), repeatable (sigma(rel) < or = 4.4%), accurate (recovery rates between 97.9 and 104.9%), and precise (intraday variation < or = 9.4%, interday variation < or = 6.2 %). For an optimum extraction of the nucleotides the chemostat samples were directly placed into hot (90 degrees C) 50% ethanol, and shaken for 10 min, followed by evaporation of the solvent and a solid phase extraction cleanup of the redissolved aqueous samples. With this method the nucleotide concentrations in hyphae from a glucose-limited chemostat culture and the respective energy charge were determined. Additionally, the effect of the time lag between sampling and extraction and the effect of a glucose pulse on nucleotide concentrations were determined.     

Oxygenation of monounsaturated fatty acids by soybean lipoxygenase-1: evidence for transient hydroperoxide formation

Soybean lipoxygenase-1 (SBLO-1) catalyzes the oxygenation of polyunsaturated fatty acids to produce conjugated diene hydroperoxides. Previous work from our laboratories has demonstrated that SBLO-1 will also catalyze the oxygenation of monounsaturated acids (Clapp, C. H., Senchak, S. E., Stover, T. J., Potter, T. C., Findeis, P. M., and Novak, M. J. (2001) Soybean Lipoxygenase-Mediated Oxygenation of Monounsaturated Fatty Acids to Enones, J. Am. Chem. Soc. 123, 747-748). Interestingly, the products are alpha,beta-unsaturated ketones rather than the expected allylic hydroperoxides. In the present work, we provide evidence that the monoolefin substrates are initially converted to allylic hydroperoxides, which are subsequently converted to the enone products. The hydroperoxide intermediates can be trapped by reduction to the corresponding allylic alcohols with glutathione peroxidase plus glutathione or with SnCl2. Under some conditions, the hydroperoxide intermediates accumulate and can be detected by HPLC and peroxide assays. Kinetics measurements at low concentrations of [1-14C]-9(Z)-octadecenoic acid indicate that oxygenation of this substrate at 25 degrees C, pH 9.0 occurs with kcat/Km = 1.6 (+/-0.1) x 10(2) M-1 s-1, which is about 105 lower than kcat/Km for oxygenation of 9(Z),12(Z)-octadecadienoic acid (linoleic acid). Comparison of the activities of 9(Z)-octadecenoic acid and 12(Z)-octadecenoic acid implies that the two double bonds of linoleic acid contribute almost equally to the C-H bond-breaking step in the normal lipoxygenase reaction. The results are consistent with the notion that SBLO-1 functionalizes substrates by a radical mechanism.     

Mice lacking the gene encoding for MMP-9 and resistance artery reactivity

OBJECTIVES: To define the link between the deletion of gene encoding for metalloproteinase 9 and resistance artery reactivity, we studied in vitro smooth muscle and endothelial cell function in response to pressure, shear stress, and pharmacological agents. BACKGROUND: Matrix metalloproteinases play a crucial role in the regulation of extracellular matrix turnover and structural artery wall remodeling. METHODS: Resistance arteries were isolated from mice lacking gene encoding for MMP-9 (KO) and their control (WT). Hemodynamic, pharmacology approaches, and Western blot analysis were used in this study. RESULTS: The measurement of blood pressure in vivo was similar in KO and WT mice. Pressure-induced myogenic tone, contractions to angiotensin-II and phenylephrine were similar in both groups. The inhibition of MMP2/9 ((2R)-2-[(4-biphenylylsulfonyl) amino]-3-phenylpropionic acid) significantly decreased myogenic tone in WT and had no effect in KO mice. Relaxation endothelium-dependent (flow-induced- dilation 41.3+/-0.6 vs. 21+/-1.6 at 10 microl/min in KO and WT mice, respectively, P<0.05) and eNOS expression were increased in KO compared to WT mice. The inhibition of eNOS with L-NAME significantly decreased endothelium response to shear stress, which was more pronounced in KO mice resistance arteries (-26.83+/-2.5 vs. -15.84+/-2.3 at 10 microl/min in KO and WT, respectively, P<0.05). However, the relaxation to exogenous nitric oxide-donor was similar in both groups. CONCLUSION: Our study provides evidence of a selective effect of MMP-9 on endothelium function. Thus, MMP-9 gene deletion specifically increased resistance artery dilation endothelium-dependent and eNOS expression. Based on our results, MMP-9 could be a potential therapeutic target in cardiovascular disease associated with resistance arteries dysfunction.