EphA receptors and ephrin-A ligands are upregulated by monocytic differentiation/maturation and promote cell adhesion and protrusion formation in HL60 monocytes

Background

Eph signaling is known to induce contrasting cell behaviors such as promoting and inhibiting cell adhesion/spreading by altering F-actin organization and influencing integrin activities. We have previously demonstrated that EphA2 stimulation by ephrin-A1 promotes cell adhesion through interaction with integrins and integrin ligands in two monocyte/macrophage cell lines. Although mature mononuclear leukocytes express several members of the EphA/ephrin-A subclass, their expression has not been examined in monocytes undergoing during differentiation and maturation.

Results

Using RT-PCR, we have shown that EphA2, ephrin-A1, and ephrin-A2 expression was upregulated in murine bone marrow mononuclear cells during monocyte maturation. Moreover, EphA2 and EphA4 expression was induced, and ephrin-A4 expression was upregulated, in a human promyelocytic leukemia cell line, HL60, along with monocyte differentiation toward the classical CD14⁺⁺CD16^? monocyte subset. Using RT-PCR and flow cytometry, we have also shown that expression levels of αL, αM, αX, and β2 integrin subunits were upregulated in HL60 cells along with monocyte differentiation while those of α4, α5, α6, and β1 subunits were unchanged. Using a cell attachment stripe assay, we have shown that stimulation by EphA as well as ephrin-A, likely promoted adhesion to an integrin ligand-coated surface in HL60 monocytes. Moreover, EphA and ephrin-A stimulation likely promoted the formation of protrusions in HL60 monocytes.

Conclusions

Notably, this study is the first analysis of EphA/ephrin-A expression during monocytic differentiation/maturation and of ephrin-A stimulation affecting monocyte adhesion to an integrin ligand-coated surface. Thus, we propose that monocyte adhesion via integrin activation and the formation of protrusions is likely promoted by stimulation of EphA as well as of ephrin-A.

     

QTL mapping for salt tolerance and domestication-related traits in Vigna marina subsp. oblonga,a halophytic species

Key message

QTL mapping in F ₂ population [ V. luteola × V. marina subsp. oblonga ] revealed that the salt tolerance in V. marina subsp. oblonga is controlled by a single major QTL.

Abstract

The habitats of beach cowpea (Vigna marina) are sandy beaches in tropical and subtropical regions. As a species that grows closest to the sea, it has potential to be a gene source for breeding salt-tolerant crops. We reported here for the first time, quantitative trait loci (QTLs) mapping for salt tolerance in V. marina. A genetic linkage map was constructed from an F₂ population of 120 plants derived from an interspecific cross between V. luteola and V. marina subsp. oblonga. The map comprised 150 SSR markers. The markers were clustered into 11 linkage groups spanning 777.6 cM in length with a mean distance between the adjacent markers of 5.59 cM. The F_2:3 population was evaluated for salt tolerance under hydroponic conditions at the seedling and developmental stages. Segregation analysis indicated that salt tolerance in V. marina is controlled by a few genes. Multiple interval mapping consistently identified one major QTL which can explain about 50 % of phenotypic variance. The flanking markers may facilitate transfer of the salt tolerance allele from V. marina subsp. oblonga into related Vigna crops. The QTL for domestication-related traits from V. marina are also discussed.     

Simultaneous stereoinversion and isomerization at the Asp-4 residue in βB2-crystallin from the aged human eye lenses

The lens proteins are composed of α-, β-, and γ-crystallins that interact with each other to maintain the transparency and refractive power of the lens. Because the lens crystallins are long-lived proteins, they undergo various post-translational modifications including racemization, isomerization, deamidation, oxidation, glycation, and truncation. In βB2-crystallin, which is the most abundant β-crystallin, the deamidation of asparagine and glutamine residues has been reported. Here, we found that the aspartyl (Asp) residue at position 4 of βB2-crystallin in the lenses of elderly human individuals undergoes a significant degree of inversion and isomerization to the biologically uncommon residue D-β-Asp. Surprisingly, the D/L ratio of β-Asp at position 4 in βB2-crystallin from elderly donors (67-77 year old) was 0.88-3.21. A D/L ratio of amino acids greater than 1.0 is defined as an inversion of configuration from the L- to D-form, rather than a racemization. These extremely high D/L ratios are equivalent to those of Asp-58 and Asp-151 (D/L ratio: 3.1 for Asp-58 and 5.7 for Asp-151) in αA-crystallin from elderly donors (~80 year old) as reported previously. Initially, we identified specific Asp residues in the β-crystallin family of proteins that undergo a high degree of inversion. These results show that the isomerization and inversion of Asp residues occurs both in the α- and β-crystallins of the lens. Inversion of these Asp residues directly affects the higher order structure of the protein. Hence, this modification may change crystallin-crystallin interactions and disrupt the function of crystallins in the lens.     

Efficient functional analysis system for cyanobacterial or plant cytochromes P450 involved in sesquiterpene biosynthesis

Tractable plasmids (pAC-Mv-based plasmids) for Escherichia coli were constructed, which carried a mevalonate-utilizing gene cluster, towards an efficient functional analysis of cytochromes P450 involved in sesquiterpene biosynthesis. They included genes coding for a series of redox partners that transfer the electrons from NAD(P)H to a P450 protein. The redox partners used were ferredoxin reductases (CamA and NsRED) and ferredoxins (CamB and NsFER), which are derived from Pseudomonas putida and cyanobacterium Nostoc sp. strain PCC 7120, respectively, as well as three higher-plant NADPH-P450 reductases, the Arabidopsis thaliana ATR2 and two corresponding enzymes derived from ginger (Zingiber officinale), named ZoRED1 and ZoRED2. We also constructed plasmids for functional analysis of two P450s, α-humulene-8-hydroxylase (CYP71BA1) from shampoo ginger (Zingiber zerumbet) and germacrene A hydroxylase (P450NS; CYP110C1) from Nostoc sp. PCC 7120, and co-transformed E. coli with each of the pAC-Mv-based plasmids. Production levels of 8-hydroxy-α-humulene with recombinant E. coli cells (for CYP71BA1) were 1.5- to 2.3-fold higher than that of a control strain without the mevalonate-pathway genes. Level of the P450NS product with the combination of NsRED and NsFER was 2.9-fold higher than that of the CamA and CamB. The predominant product of P450NS was identified as 1,2,3,5,6,7,8,8a-octahydro-6-isopropenyl-4,8a-dimethylnaphth-1-ol with NMR analyses.     

Fission yeast Pot1 and RecQ helicase are required for efficient chromosome segregation

Pot1 is a single-stranded telomere-binding protein that is conserved from fission yeast to mammals. Deletion of Schizosaccharomyces pombe pot1(+) causes immediate telomere loss. S. pombe Rqh1 is a homolog of the human RecQ helicase WRN, which plays essential roles in the maintenance of genomic stability. Here, we demonstrate that a pot1Δ rqh1-hd (helicase-dead) double mutant maintains telomeres that are dependent on Rad51-mediated homologous recombination. Interestingly, the pot1Δ rqh1-hd double mutant displays a "cut" (cell untimely torn) phenotype and is sensitive to the antimicrotubule drug thiabendazole (TBZ). Moreover, the chromosome ends of the double mutant do not enter the pulsed-field electrophoresis gel. These results suggest that the entangled chromosome ends in the pot1Δ rqh1-hd double mutant inhibit chromosome segregation, signifying that Pot1 and Rqh1 are required for efficient chromosome segregation. We also found that POT1 knockdown, WRN-deficient human cells are sensitive to the antimicrotubule drug vinblastine, implying that some of the functions of S. pombe Pot1 and Rqh1 may be conserved in their respective human counterparts POT1 and WRN.     

PARK2/Parkin-mediated mitochondrial clearance contributes to proteasome activation during slow-twitch muscle atrophy via NFE2L1 nuclear translocation

Skeletal muscle atrophy is thought to result from hyperactivation of intracellular protein degradation pathways, including autophagy and the ubiquitin–proteasome system. However, the precise contributions of these pathways to muscle atrophy are unclear. Here, we show that an autophagy deficiency in denervated slow-twitch soleus muscles delayed skeletal muscle atrophy, reduced mitochondrial activity, and induced oxidative stress and accumulation of PARK2/Parkin, which participates in mitochondrial quality control (PARK2-mediated mitophagy), in mitochondria. Soleus muscles from denervated Park2 knockout mice also showed resistance to denervation, reduced mitochondrial activities, and increased oxidative stress. In both autophagy-deficient and Park2-deficient soleus muscles, denervation caused the accumulation of polyubiquitinated proteins. Denervation induced proteasomal activation via NFE2L1 nuclear translocation in control mice, whereas it had little effect in autophagy-deficient and Park2-deficient mice. These results suggest that PARK2-mediated mitophagy plays an essential role in the activation of proteasomes during denervation atrophy in slow-twitch muscles.     

Structure of extrachromosomal circular DNAs excised from T-cell antigen receptor alpha and delta-chain loci

Small polydisperse circular (spc) DNA was isolated from mouse thymocytes, fragmented by HindIII digestion and cloned into the vector. Sixty DNA clones were randomly selected from the 10,400 phage library. The average size of insert was one-fifth of the original circular molecule. Twenty spc-DNA clones were homologous to DNA probes derived from T-cell antigen receptor (TCR) alpha-chain loci. We have characterized nine clones by DNA sequencing; they contain new germline sequences of the TCR alpha-chain variable (V alpha) and joining (J alpha) gene segments and the products out of the recombination of a V alpha with a J alpha gene segment. An additional four spc-DNA clones carried a new rearranging gene of the TCR delta-chain that is located between V alpha and J alpha genes. At least nine of 60 DNA clones carried the recombination junction of a heptamer-heptamer head-to-head structure expected from an excised product of V-J joining. This shows that most extrachromosomal circular DNAs in the thymus are formed by a sequence-dependent recombination mechanism. We suggest that a functional T-cell receptor V alpha gene can be constructed by somatic random rearrangements through successive looping-out, excision and deletion.     

Isolation and functional identification of a novel cDNA for astaxanthin biosynthesis from Haematococcus pluvialis,and astaxanthin synthesis in Escherichia coli

We succeeded in isolating a novel cDNA involved in astaxanthin biosynthesis from the green alga Haematococcus pluvialis, by an expression cloning method using an Escherichia coli transformant as a host that synthesizes -carotene due to the Erwinia uredovora carotenoid biosynthesis genes. The cloned cDNA was shown to encode a novel enzyme, -carotene ketolase (-carotene oxygenase), which converted -carotene to canthaxanthin via echinenone, through chromatographic and spectroscopic analysis of the pigments accumulated in an E. coli transformant. This indicates that the encoded enzyme is responsible for the direct conversion of methylene to keto groups, a mechanism that usually requires two different enzymatic reactions proceeding via a hydroxy intermediate. Northern blot analysis showed that the mRNA was synthesized only in the cyst cells of H. pluvialis. E. coli carrying the H. pluvialis cDNA and the E. uredovora genes required for zeaxanthin biosynthesis was also found to synthesize astaxanthin (3S, 3S), which was identified after purification by a variety of spectroscopic methods.