Molecular cloning of a pair of human pepsinogen A genes which differ by a Glu→Lys mutation in the activation peptide

Summary Three human cosmid clones containing pepsinogen A (PGA) encoding sequences were isolated from a genomic bank derived from a single individual. One cosmid contains two PGA genes in tandem in a head-to-tail orientation, while the other two cosmids each contain a single PGA gene. The three cosmids were characterized by restriction mapping and sequence analysis (exons 1 and 2 and flanking regions). As judged from these data, three of the four PGA genes isolated appear to be nearly identical, but one of the tandem genes is clearly different from the other genes. The first exon of all four genes codes for the same amino acid sequence. However, in the second exon of one of the tandem genes we found a nucleotide substitution giving rise to a GluLys substitution of the 43rd amino acid residue of the activation peptide, leading to a charge difference of the corresponding isozymogens. The presence of two distinct PGA genes in the isolated gene pair conclusively proves the multigene structure of the PGA system. These genes might be responsible for at least part of the electrophoretic polymorphism at the protein level.     

UsingGCN4as a Reporter of eIF2α Phosphorylation and Translational Regulation in Yeast

Molecular genetic analyses in yeast are a powerful method to study gene regulation. Conservation of the mechanism and regulation of protein synthesis between yeast and mammalian cells makes yeast a good model system for the analysis of translation. One of the most common mechanisms of translational regulation in mammalian cells is the phosphorylation of serine-51 on the α subunit of the translation initiation factor eIF2, which causes an inhibition of general translation. In contrast, in the yeastSaccharomyces cerevisiaephosphorylation of eIF2α on serine-51 by theGCN2protein kinase mediates the translational induction ofGCN4expression. The unique structure of theGCN4mRNA makesGCN4expression especially sensitive to eIF2α phosphorylation, and the simple microbiological tests developed in yeast to analyzeGCN4expression serve as good reporters of eIF2α phosphorylation. It is relatively simple to express heterologous proteins in yeast, and it has been shown that the mammalian eIF2α kinases will functionally substitute forGCN2.Structure–function analyses of translation factors or translational regulators can also be performed by assaying for effects on general andGCN4-specific translation. Three tests can be used to study eIF2α phosphorylation and/or translational activity in yeast. First, general translation can be monitored by simple growth tests, whileGCN4expression can be analyzed using sensitive replica-plating tests. Second,GCN4translation can be quantitated by measuring expression fromGCN4–lacZreporter constructs. Finally, isoelectric focusing gels can be used to directly monitorin vivophosphorylation of eIF2α in yeast.     

Regulation of 11β-hydroxysteroid dehydrogenase 1 and 2 by IGF-1 in mice

Two isoforms of 11β-hydroxysteroid dehydrogenase (11β-HSD1 and 11β-HSD2) play an important role in regulation of glucocorticoid corticosterone (CORT, the active form in rodents) by the interconversion between CORT and 11-dehydrocorticosterone (11DHC, the biologically inert form). 11β-HSD1 is an NADP+/NADPH-dependent oxidoreductase which is mainly expressed in liver and kidney, while 11β-HSD2 is an NAD+-dependent oxidase which is predominantly expressed in kidney. The regulation of 11β-HSD1 and 11β-HSD2 mRNA (Hsd11b1 and Hsd11b2) levels and their activities by IGF-1 was performed in liver, kidney, and testis of IGF-1 knockout male mice. Real-time PCR showed that Hsd11b1 in liver was decreased while Hsd11b2 mRNA level was decreased in kidney of IGF-1 null mice. 11β-HSD1 and 11β-HSD2 activities fluctuated with the changes of their respective Hsd11b1 or Hsd11b2 mRNA levels. In conclusion, IGF-I tissue-specifically regulates Hsd11b1 and Hsd11b2 expression.     

Isolation of SPO12–1 and SPO13–1 from a Natural Variant of Yeast That Undergoes a Single Meiotic Division

ATCC4117 is a strain of S. cerevisiae that undergoes a single nuclear division during sporulation to produce asci containing two diploid ascospores (Grewal and Miller 1972). All clones derived from these spores are sporulation-capable and, like the parental strain, form two-spored asci. In this paper, we describe the genetic analysis of ATCC4117. In tetraploid hybrids of vegetative cells of the ATCC4117 diploid and a/a or α/α diploids, the production of two-spored asci is recessive. From these tetraploids, we have isolated two recessive alleles, designated spo12–1 and spo13–1, each of which alone results in the production of asci with two diploid or near-diploid spores. These alleles are unlinked and segregate as single nuclear genes. spo12–1 is approximately 22 cM from its centromere; spo13–1 has been localized to within 1 cM of arg4 on chromosome VIII. This analysis also revealed that ATCC4117 carries a diploidization gene allelic to or closely linked to HO, modifiers that reduce the number of haploid spores per ascus and alleles affecting the total level of sporulation.     

Coordinate Regulation of TPL-2 and NF-κB Signaling in Macrophages by NF-κB1 p105

The role of IκB kinase (IKK)-induced proteolysis of NF-κB1 p105 in innate immune signaling was investigated using macrophages from Nfkb1(SSAA/SSAA) mice, in which the IKK target serines on p105 are mutated to alanines. We found that the IKK/p105 signaling pathway was essential for TPL-2 kinase activation of extracellular signal-regulated kinase (ERK) mitogen-activate protein (MAP) kinase and modulated the activation of NF-κB. The Nfkb1(SSAA) mutation prevented the agonist-induced release of TPL-2 from its inhibitor p105, which blocked activation of ERK by lipopolysaccharide (LPS), tumor necrosis factor (TNF), CpG, tripalmitoyl-Cys-Ser-Lys (Pam(3)CSK), poly(I · C), flagellin, and R848. The Nfkb1(SSAA) mutation also prevented LPS-induced processing of p105 to p50 and reduced p50 levels, in addition to decreasing the nuclear translocation of RelA and cRel. Reduced p50 in Nfkb1(SSAA/SSAA) macrophages significantly decreased LPS induction of the IκBζ-regulated Il6 and Csf2 genes. LPS upregulation of Il12a and Il12b mRNAs was also impaired although specific blockade of TPL-2 signaling increased expression of these genes at late time points. Activation of TPL-2/ERK signaling by IKK-induced p105 proteolysis, therefore, induced a negative feedback loop to downregulate NF-κB-dependent expression of the proinflammatory cytokine interleukin-12 (IL-12). Unexpectedly, TPL-2 promoted soluble TNF production independently of IKK-induced p105 phosphorylation and its ability to activate ERK, which has important implications for the development of anti-inflammatory drugs targeting TPL-2.     

Regulation of plankton by omnivore cyprinids in a shallow lake in the Kis–Balaton Reservoir System

Hydrobiologia - A long-term food web manipulation experiment was started in 1999 with monitoring in the eutrophic shallow Major Lake (area 11 ha, mean depth 1.1 m). In 2000, studies were continued...     

Cell Cycle– and Chaperone-Mediated Regulation of H3K56ac Incorporation in Yeast

Acetylation of histone H3 lysine 56 is a covalent modification best known as a mark of newly replicated chromatin, but it has also been linked to replication-independent histone replacement. Here, we measured H3K56ac levels at single-nucleosome resolution in asynchronously growing yeast cultures, as well as in yeast proceeding synchronously through the cell cycle. We developed a quantitative model of H3K56ac kinetics, which shows that H3K56ac is largely explained by the genomic replication timing and the turnover rate of each nucleosome, suggesting that cell cycle profiles of H3K56ac should reveal most first-time nucleosome incorporation events. However, since the deacetylases Hst3/4 prevent use of H3K56ac as a marker for histone deposition during M phase, we also directly measured M phase histone replacement rates. We report a global decrease in turnover rates during M phase and a further specific decrease in turnover at several early origins of replication, which switch from rapidly replaced in G1 phase to stably bound during M phase. Finally, by measuring H3 replacement in yeast deleted for the H3K56 acetyltransferase Rtt109 and its two co-chaperones Asf1 and Vps75, we find evidence that Rtt109 and Asf1 preferentially enhance histone replacement at rapidly replaced nucleosomes, whereas Vps75 appears to inhibit histone turnover at those loci. These results provide a broad perspective on histone replacement/incorporation throughout the cell cycle and suggest that H3K56 acetylation provides a positive-feedback loop by which replacement of a nucleosome enhances subsequent replacement at the same location.     

Regulation of BCRP (ABCG2) and P-Glycoprotein (ABCB1) by Cytokines in a Model of the Human Blood–Brain Barrier

Brain capillary endothelial cells form the blood–brain barrier (BBB), a highly selective permeability membrane between the blood and the brain. Besides tight junctions that prevent small hydrophilic compounds from passive diffusion into the brain tissue, the endothelial cells express different families of drug efflux transport proteins that limit the amount of substances penetrating the brain. Two prominent efflux transporters are the breast cancer resistance protein and P-glycoprotein (P-gp). During inflammatory reactions, which can be associated with an altered BBB, pro-inflammatory cytokines are present in the systemic circulation. We, therefore, investigated the effect of the pro-inflammatory cytokines interleukin-1β (IL-1β), interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) on the expression and activity of BCRP and P-gp in the human hCMEC/D3 cell line. BCRP mRNA levels were significantly reduced by IL-1β, IL-6 and TNF-α. The strongest BCRP suppression at the protein level was observed after IL-1β treatment. IL-1β, IL-6 and TNF-α also significantly reduced the BCRP activity as assessed by mitoxantrone uptake experiments. P-gp mRNA levels were slightly reduced by IL-6, but significantly increased after TNF-α treatment. TNF-α also increased protein expression of P-gp but the uptake of the P-gp substrate rhodamine 123 was not affected by any of the cytokines. This in vitro study indicates that expression levels and activity of BCRP, and P-gp at the BBB may be altered by acute inflammation, possibly affecting the penetration of their substrates into the brain.     

Importance of Bacteroidetes in host–microbe interactions and ecosystem functioning

Bacteroidetes are prevalent in soil ecosystems and are associated with various eukaryotic hosts, including plants, animals, and humans. The ubiquity and diversity of Bacteroidetes exemplify their impressive versatility in niche adaptation and genomic plasticity. Over the past decade, a wealth of knowledge has been obtained on the metabolic functions of clinically relevant Bacteroidetes, but much less attention has been given to Bacteroidetes living in close association with plants. To improve our understanding of the functional roles of Bacteroidetes for plants and other hosts, we review the current knowledge of their taxonomy and ecology, in particular their roles in nutrient cycling and host fitness. We highlight their environmental distribution, stress resilience, genomic diversity, and functional importance in diverse ecosystems, including, but not limited to, plant-associated microbiomes.     

Regulation of PTEN degradation and NEDD4–1 E3 ligase activity by Numb

The critical tumor suppressor PTEN is regulated by numerous post-translational modifications including phosphorylation, acetylation and ubiquitination. Ubiquitination of PTEN was reported to control both PTEN stability and nuclear localization. Notably, the HECT E3-ligase NEDD4–1 was identified as the ubiquitin ligase for PTEN, mediating its degradation and down-stream events. However, the mechanisms how NEDD4–1 is regulated by up-stream signaling pathways or interaction with other proteins in promoting PTEN degradation remain largely unclear. In the present study, we identified that the adaptor protein Numb, which is demonstrated to be a novel binding partner of NEDD4–1, plays important roles in controlling PTEN ubiquitination through regulating NEDD4–1 activity and the association between PTEN and NEDD4–1. Furthermore, we provided data to show that Numb regulates cell proliferation and glucose metabolism in a PTEN-dependent manner. Overall, our study revealed a novel regulation of the well-documented NEDD4–1/PTEN pathway and its oncogenic behavior.     

Neuronal IP3 3-Kinase is an F-actin–bundling Protein: Role in Dendritic Targeting and Regulation of Spine Morphology

The actin microstructure in dendritic spines is involved in synaptic plasticity. Inositol trisphosphate 3-kinase A (ITPKA) terminates Ins(1,4,5)P₃ signals emanating from spines and also binds filamentous actin (F-actin) through its amino terminal region (amino acids 1-66, N66). Here we investigated how ITPKA, independent of its kinase activity, regulates dendritic spine F-actin microstructure. We show that the N66 region of the protein mediates F-actin bundling. An N66 fusion protein bundled F-actin in vitro, and the bundling involved N66 dimerization. By mutagenesis we identified a point mutation in a predicted helical region that eliminated both F-actin binding and bundling, rendering the enzyme cytosolic. A fusion protein containing a minimal helical region (amino acids 9-52, N9-52) bound F-actin in vitro and in cells, but had lower affinity. In hippocampal neurons, GFP-tagged N66 expression was highly polarized, with targeting of the enzyme predominantly to spines. By contrast, N9-52-GFP expression occurred in actin-rich structures in dendrites and growth cones. Expression of N66-GFP tripled the length of dendritic protrusions, induced longer dendritic spine necks, and induced polarized actin motility in time-lapse assays. These results suggest that, in addition to its ability to regulate intracellular Ca²⁺ via Ins(1,4,5)P₃ metabolism, ITPKA regulates structural plasticity.     

Regulation of seedling growth by ethylene and the ethylene–auxin crosstalk

Planta - This review highlights that the auxin gradient, established by local auxin biosynthesis and transport, can be controlled by ethylene, and steers seedling growth. A better understanding of...     

Characterization of immunoreactive acetyl–Ser–Asp–Lys–Pro in human plasma and urine by liquid chromatography–electrospray mass spectrometry

The tetrapeptide AcSDKP, a natural and specific substrate of angiotensin I-converting enzyme (ACE), is a negative regulator of hematopoiesis. AcSDKP has been measured in various biological media using an enzyme immunoassay (EIA), but its presence in human plasma and urine has not been formally established. By using immunoaffinity extraction and liquid chromatography–electrospray mass spectrometry, we demonstrate that AcSDKP-like immunoreactivity measured with EIA in plasma and urine samples from untreated, captopril- (an ACE inhibitor) and AcSDKP-treated subjects corresponds to AcSDKP. The present study confirms that AcSDKP is naturally present in human plasma and urine and that EIA is reliable for its measurement in such media.     

Differential regulation of p53 function by the N-terminal ΔNp53 and Δ113p53 isoforms in zebrafish embryos

Background  

The p53 protein family coordinates stress responses of cells and organisms. Alternative promoter usage and/or splicing of p53 mRNA gives rise to at least nine mammalian p53 proteins with distinct N- and C-termini which are differentially expressed in normal and malignant cells. The human N-terminal p53 variants contain either the full-length (FL), or a truncated (ΔN/Δ40) or no transactivation domain (Δ133) altogether. The functional consequences of coexpression of the different p53 isoforms are poorly defined. Here we investigated functional aspects of the zebrafish ΔNp53 ortholog in the context of FLp53 and the zebrafish Δ133p53 ortholog (Δ113p53) coexpressed in the developing embryo.     

Regulation of ClC-1 and KATP channels in action potential–firing fast-twitch muscle fibers

Action potential (AP) excitation requires a transient dominance of depolarizing membrane currents over the repolarizing membrane currents that stabilize the resting membrane potential. Such stabilizing currents, in turn, depend on passive membrane conductance (G_m), which in skeletal muscle fibers covers membrane conductances for K⁺ (G_K) and Cl⁻ (G_Cl). Myotonic disorders and studies with metabolically poisoned muscle have revealed capacities of G_K and G_Cl to inversely interfere with muscle excitability. However, whether regulation of G_K and G_Cl occur in AP-firing muscle under normal physiological conditions is unknown. This study establishes a technique that allows the determination of G_Cl and G_K with a temporal resolution of seconds in AP-firing muscle fibers. With this approach, we have identified and quantified a biphasic regulation of G_m in active fast-twitch extensor digitorum longus fibers of the rat. Thus, at the onset of AP firing, a reduction in G_Cl of ∼70% caused G_m to decline by ∼55% in a manner that is well described by a single exponential function characterized by a time constant of ∼200 APs (phase 1). When stimulation was continued beyond ∼1,800 APs, synchronized elevations in G_K (∼14-fold) and G_Cl (∼3-fold) caused G_m to rise sigmoidally to ∼400% of its level before AP firing (phase 2). Phase 2 was often associated with a failure to excite APs. When AP firing was ceased during phase 2, G_m recovered to its level before AP firing in ∼1 min. Experiments with glibenclamide (K_ATP channel inhibitor) and 9-anthracene carboxylic acid (ClC-1 Cl⁻ channel inhibitor) revealed that the decreased G_m during phase 1 reflected ClC-1 channel inhibition, whereas the massively elevated G_m during phase 2 reflected synchronized openings of ClC-1 and K_ATP channels. In conclusion, G_Cl and G_K are acutely regulated in AP-firing fast-twitch muscle fibers. Such regulation may contribute to the physiological control of excitability in active muscle.