An overview on chlorophylls and quinones in the photosystem I-type reaction centers

Minor but key chlorophylls (Chls) and quinones in photosystem (PS) I-type reaction centers (RCs) are overviewed in regard to their molecular structures. In the PS I-type RCs, the prime-type chlorophylls, namely, bacteriochlorophyll (BChl) a′ in green sulfur bacteria, BChl g′ in heliobacteria, Chl a′ in Chl a-type PS I, and Chl d′ in Chl d-type PS I, function as the special pairs, either as homodimers, (BChl a′)₂ and (BChl g′)₂ in anoxygenic organisms, or heterodimers, Chl a/a′ and Chl d/d′ in oxygenic photosynthesis. Conversions of BChl g to Chl a and Chl a to Chl d take place spontaneously under mild condition in vitro. The primary electron acceptors, A ₀, are Chl a-derivatives even in anoxygenic PS I-type RCs. The secondary electron acceptors are naphthoquinones, whereas the side chains may have been modified after the birth of cyanobacteria, leading to succession from menaquinone to phylloquinone in oxygenic PS I.     

Ehd4 is required to attain normal prepubertal testis size but dispensable for fertility in male mice

The four highly homologous members of the C‐terminal EH domain‐containing (EHD) protein family (EHD1‐4) regulate endocytic recycling. To delineate the role of EHD4 in normal physiology and development, mice with a conditional knockout of the Ehd4 gene were generated. PCR of genomic DNA and Western blotting of organ lysates from Ehd4^−/− mice confirmed EHD4 deletion. Ehd4^−/− mice were viable and born at expected Mendelian ratios; however, males showed a 50% reduction in testis weight, obvious from postnatal day 31. An early (Day 10) increase in germ cell proliferation and apoptosis and a later increase in apoptosis (Day 31) were seen in the Ehd4^−/− testis. Other defects included a progressive reduction in seminiferous tubule diameter, dysregulation of seminiferous epithelium, and head abnormalities in elongated spermatids. As a consequence, lower sperm counts and reduced fertility were observed in Ehd4^−/− males. Interestingly, EHD protein expression was seen to be temporally regulated in the testis and EHD4 levels peaked between days 10 and 15. In the adult testis, EHD4 was highly expressed in primary spermatocytes and EHD4 deletion altered the levels of other EHD proteins in an age‐dependent manner. We conclude that high levels of EHD1 in the adult Ehd4^−/− testis functionally compensate for lack of EHD4 and prevents the development of severe fertility defects. Our results suggest a role for EHD4 in the proper development of postmitotic and postmeiotic germ cells and implicate EHD protein‐mediated endocytic recycling as an important process in germ cell development and testis function. genesis 48:328–342, 2010. © 2010 Wiley‐Liss, Inc.     

The plant growth-promoting fungus Penicillium simplicissimum GP17-2 induces resistance in Arabidopsis thaliana by activation of multiple defense signals

Arabidopsis thaliana grown in soil amended with barley grain inocula of Penicillium simplicissimum GP17-2 or receiving root treatment with its culture filtrate (CF) exhibited clear resistance to Pseudomonas syringae pv. tomato DC3000 (Pst). To assess the contribution of different defense pathways, Arabidopsis genotypes implicated in salicylic acid (SA) signaling expressing the NahG transgene or carrying disruption in NPR1 (npr1), jasmonic acid (JA) signaling (jar1) and ethylene (ET) signaling (ein2) were tested. All genotypes screened were protected by GP17-2 or its CF. However, the level of protection was significantly lower in NahG and npr1 plants than it was in similarly treated wild-type plants, indicating that the SA signaling pathway makes a minor contribution to the GP17-2-mediated resistance and is insufficient for a full response. Examination of local and systemic gene expression revealed that GP17-2 and its CF modulate the expression of genes involved in both the SA and JA/ET signaling pathways. Subsequent challenge of GP17-2-colonized plants with Pst was accompanied by direct activation of SA-inducible PR-2 and PR-5 genes as well as potentiated expression of the JA-inducible Vsp gene. In contrast, CF-treated plants infected with Pst exhibited elevated expression of most defense-related genes (PR-1, PR-2, PR-5, PDF1.2 and Hel) studied. Moreover, an initial elevation of SA responses was followed by late induction of JA responses during Pst infection of induced systemic resistance (ISR)-expressing plants. In conclusion, we hypothesize the involvement of multiple defense mechanisms leading to an ISR of Arabidopsis by GP17-2.     

Progesterone: its occurrence in plants and involvement in plant growth

Progesterone is a mammalian gonadal hormone. In the current study, we identified and quantified progesterone in a range of higher plants by using GC-MS and examined its effects on the vegetative growth of plants. The growth of Arabidopsis (Arabidopsis thaliana) seedlings was promoted by progesterone at low concentrations but suppressed at higher concentrations under both light and dark growth conditions. The growth of the gibberellin-deficient mutant lh of pea (Pisum sativum) was also promoted by progesterone. An earlier study demonstrated that progesterone binds to MEMBRANE STEROID BINDING PROTEIN 1 (MSBP1) of Arabidopsis. In this work, we cloned the homologous genes of Arabidopsis, MSBP2 and STEROID BINDING PROTEIN (SBP), as well as of rice (Oryza sativa), OsMSBP1, OsMSBP2 and OsSBP and examined their expression in plant tissues. All of these genes, except OsMSBP1, were expressed abundantly in plant tissues. The roles of progesterone in plant growth were also discussed.     

Structural comparison of fucosylated and nonfucosylated Fc fragments of human immunoglobulin G1

Removal of the fucose residue from the oligosaccharides attached to Asn297 of human immunoglobulin G1 (IgG1) results in a significant enhancement of antibody-dependent cellular cytotoxicity (ADCC) via improved IgG1 binding to Fcgamma receptor IIIa. To provide structural insight into the mechanisms of affinity enhancement, we determined the crystal structure of the nonfucosylated Fc fragment and compared it with that of fucosylated Fc. The overall conformations of the fucosylated and nonfucosylated Fc fragments were similar except for hydration mode around Tyr296. Stable-isotope-assisted NMR analyses confirmed the similarity of the overall structures between fucosylated and nonfucosylated Fc fragments in solution. These data suggest that the glycoform-dependent ADCC enhancement is attributed to a subtle conformational alteration in a limited region of IgG1-Fc. Furthermore, the electron density maps revealed that the traces between Asp280 and Asn297 of our fucosylated and nonfucosylated Fc crystals were both different from that in previously reported isomorphous Fc crystals.     

Role of spinal voltage-dependent calcium channel alpha 2 delta-1 subunit in the expression of a neuropathic pain-like state in mice

The present study was undertaken to investigate the role of spinal voltage-dependent calcium channel alpha(2)delta-1 subunit in the expression of a neuropathic pain-like state induced by partial sciatic nerve ligation in mice. In cultured spinal neurons, gabapentin (GBP), which displays the inhibitory effect of alpha(2)delta-1 subunit, suppressed the extracellular Ca(2+) influx induced by KCl, whereas it failed to inhibit the intracellular Ca(2+) release induced by inositol-1,4,5-triphosphate. Seven days after sciatic nerve ligation, the protein level of alpha(2)delta-1 subunit in the ipsilateral spinal cord was clearly increased compared to that observed in sham-operated mice. In addition, the mRNA level of alpha(2)delta-1 subunit was significantly increased in the dorsal root ganglion, but not in the spinal cord, of nerve-ligated mice. Under these conditions, a marked decrease in the latency of paw-withdrawal against a thermal stimulation and tactile stimulation, induced by sciatic nerve ligation was abolished by repeated intrathecal (i.t.) treatment with GBP. Additionally, the persistent reduction in the nociceptive threshold by i.t. treatment with GBP at the early stage of the neuropathic pain-like state was maintained for 7 days even after GBP withdrawal. It is of interest to note that a single i.t. post-injection of GBP showed a marked and transient inhibitory effect on the developed neuropathic pain-like state, whereas repeated i.t. post-treatment with GBP produced a persistent inhibitory effect during the treatment. In conclusion, we propose here that the neuropathic pain-like state with sciatic nerve ligation is associated with the increased level of the alpha(2)delta-1 subunit of Ca(2+) channels at the sensory nerve terminal in the spinal dorsal horn of mice. Furthermore, the present data provide evidence that the neuropathic pain may be effectively controlled by repeated treatment with GBP at the early stage.     

Tom20 recognizes mitochondrial presequences through dynamic equilibrium among multiple bound states

Most mitochondrial proteins are synthesized in the cytosol and imported into mitochondria. The N-terminal presequences of mitochondrial-precursor proteins contain a diverse consensus motif (phi chi chi phi phi, phi is hydrophobic and chi is any amino acid), which is recognized by the Tom20 protein on the mitochondrial surface. To reveal the structural basis of the broad selectivity of Tom20, the Tom20-presequence complex was crystallized. Tethering a presequence peptide to Tom20 through a disulfide bond was essential for crystallization. Unexpectedly, the two crystals with different linker designs provided unique relative orientations of the presequence with respect to Tom20, and neither configuration could fully account for the hydrophobic preference at the three hydrophobic positions of the consensus motif. We propose the existence of a dynamic equilibrium in solution among multiple states including the two bound states. In accordance, NMR 15N relaxation analyses suggested motion on a sub-millisecond timescale at the Tom20-presequence interface. We suggest that the dynamic, multiple-mode interaction is the molecular mechanism facilitating the broadly selective specificity of the Tom20 receptor toward diverse mitochondrial presequences.     

Cyclic peptide analysis of the biologically active loop region in the laminin alpha3 chain LG4 module demonstrates the importance of peptide conformation on biological activity

The laminin alpha3 chain LG4 module (alpha3LG4 module) has cell adhesion, heparin binding, migration, and neurite outgrowth activities. The LG4 module consists of a 14-stranded beta-sheet (A-N) sandwich structure. Previously, we identified the A3G756 sequence (KNSFMALYLSKGRLVFALG in the human laminin alpha3 chain 1411-1429) as a biologically active site in the alpha3LG4 module. The A3G756 sequence is located on the E and F strands based on a crystal structure-based sequence alignment. The Lys1421 and Arg1423 residues, critical amino acids for the biological activity of A3G756, are located on the E-F connecting loop region as a KGR sequence. In this study, we focused on the KGR sequence and investigated the structural requirements of the E-F connecting loop region in the alpha3LG4 module. We synthesized three linear peptides containing the KGR sequence at the middle and the N and C termini and also prepared three cyclic analogues corresponding to the linear peptides. cyclo-hEF3A (CLYLSKGRLVFAC), which is a cyclic peptide containing the KGR sequence at the middle, showed the strongest inhibitory effect on both the heparin binding and the cell attachment to the recombinant alpha3LG4 module protein. The cyclo-hEF3A peptide was more active for syndecan-4 binding and neurite outgrowth than the linear form. Furthermore, we found that the structure of cyclo-hEF3A is similar to that of the connecting E-F loop region in human laminin alpha3LG4 module by structural analysis using molecular dynamics simulations. These results suggest that the loop structure of the E-F connecting region of the alpha3LG4 module is important for its biological activities. The cyclo-hEF3A peptide may be useful for the development of therapeutic reagents especially for wound healing and nerve regeneration.     

Parasporin-2Ab,a Newly Isolated Cytotoxic Crystal Protein from <Emphasis Type="Italic">Bacillus thuringiensis</Emphasis>

A novel crystal protein that exhibited potent cytotoxicity against human leukemic T-cells was cloned from the Bacillus thuringiensis TK-E6 strain. The protein, designated as parasporin-2Ab (PS2Ab), was a polypeptide of 304 amino acid residues with a predicted molecular weight of 33,017. The deduced amino acid sequence of PS2Ab showed significant homology (84% identitiy) to parasporin-2Aa (PS2Aa) from the B. thuringiensis A1547 strain. Upon processing of PS2Ab with proteinase K, the active form of 29 kDa was produced. The activated PS2Ab showed potent cytotoxicity against MOLT-4 and Jurkat cells and the EC₅₀ values were estimated as 0.545 and 0.745 ng/mL, respectively. The cytotoxicity of PS2Ab was significantly higher than that of PS2Aa reported elsewhere. Although both cytotoxins were structurally related, it was thought that the minor differences found were responsible for the different cytotoxicities of PS2Ab and PS2Aa.