Neutral Cysteine Protease Bleomycin Hydrolase Is Essential for the Breakdown of Deiminated Filaggrin into Amino Acids

Filaggrin is a component of the cornified cell envelope and the precursor of free amino acids acting as a natural moisturizing factor in the stratum corneum. Deimination is critical for the degradation of filaggrin into free amino acids. In this study, we tried to identify the enzyme(s) responsible for the cleavage of deiminated filaggrin in vitro. First, we investigated citrulline aminopeptidase activity in the extract of newborn rat epidermis by double layer fluorescent zymography and detected strong activity at neutral pH. Monitoring the citrulline-releasing activity, we purified an enzyme of 280 kDa, comprised of six identical subunits of 48 kDa. The NH₂ terminus of representative tryptic peptides perfectly matched the sequence of rat bleomycin hydrolase (BH). The enzyme released various amino acids except Pro from β-naphthylamide derivatives and hydrolyzed citrulline-β-naphthylamide most effectively. Thus, to break down deiminated filaggrin, another protease would be required. Among proteases tested, calpain I degraded the deiminated filaggrin effectively into many peptides of different mass on the matrix-assisted laser desorption/ionization-time of flight mass spectrum. We confirmed that various amino acids including citrulline were released by BH from those peptides. On the other hand, caspase 14 degraded deiminated filaggrin into a few peptides of limited mass. Immunohistochemical analysis of normal human skin revealed co-localization of BH and filaggrin in the granular layer. Collectively, our results suggest that BH is essential for the synthesis of natural moisturizing factors and that calpain I would play a role as an upstream protease in the degradation of filaggrin.The mammalian epidermal keratinocytes arise from proliferating basal cells and move outward through a series of distinct differentiation events to form the stratum corneum (1, 2). During this progressive epidermal differentiation, keratinocytes express different proteins such as keratins, profilaggrin/filaggrin, involucrin, small proline-rich proteins, loricrin, cystatin A, and elafin, which form the cornified envelope of mature corneocytes (3–7). Profilaggrin is synthesized as a large, extremely insoluble phosphoprotein that consists of a unique NH₂-terminal Ca²⁺-binding protein of the S-100 family, linked to 10–20 tandem filaggrin monomer repeats (8–10). Each individual filaggrin repeat is completely removed by proteolysis to generate the mature filaggrin monomer (a molecular mass of 37 kDa in human). Then, filaggrin is completely degraded in the uppermost layer of the stratum corneum to produce a mixture of free and modified hygroscopic amino acids that are important for maintaining epidermal hydration (2, 11–13). In addition, a number of proteins are subjected to various post-translational modifications such as disulfide bonding, N-(γ-glutamyl)-lysine isopeptide cross-linking, and deimination during the terminal differentiation of epidermal keratinocytes (4, 6, 14, 15). Deimination is catalyzed by peptidylarginine deiminase (PAD),² which converts arginine to citrulline in proteins (17–19). The modification seems essential for the processing into free amino acids including citrulline.Several proteases reportedly participate in the processing of profilaggrin. Furin, a member of the proprotein convertase family, has been proposed to cleave the NH₂ terminus of profilaggrin, facilitating the release of the NH₂-terminal S-100 protein (20, 21). In contrast, calpain I and profilaggrin endopeptidase I (PEP-I) were implicated in the processing of the linker regions between the filaggrin monomer repeats to generate the filaggrin monomer (22–25). Recently, significant results regarding the conversion of profilaggrin to filaggrin have been obtained with the knock-out of matriptase/MT-SP1, prostasin/channel-activating serine protease 1/Prss 8, and caspase 14 in mice (26–28). These proteases were a key component of the profilaggrin-processing pathway in terminal epidermal differentiation. However, although the signal initiating the degradation of profilaggrin at a defined stage of the maturation of the stratum corneum was found to be the water gradient within the stratum corneum itself (11), the proteases for the processing of filaggrin and/or the deiminated form into peptides following the breakdown of these peptides to amino acids including citrulline remain unknown.In this study, we have purified a novel aminopeptidase using a deiminated substrate from rat skin homogenate and identified it as a neutral cysteine protease, bleomycin hydrolase (BH). Furthermore, we investigated the processing of the deiminated filaggrin by calpain I or caspase 14. Based on these results, we proposed that calpain I participated preferentially in the processing of deiminated filaggrin into peptides and then BH appeared essential for the breakdown of the peptides into amino acids.     

Interaction of the Aromatics Tyr-72/Trp-288 in the Interface of the Extracellular and Transmembrane Domains Is Essential for Proton Gating of Acid-sensing Ion Channels

Acid-sensing ion channels are proton-activated ion channels expressed in the nervous system. They belong to the family of ENaC/Degenerins whose members share a conserved structure but are activated by widely diverse stimuli. We show that interaction of two aromatic residues, Tyr-72, located immediately after the first transmembrane segment, and Trp-288, located at the tip of a loop of the extracellular domain directed toward the first transmembrane segment, is essential for proton activation of the acid-sensing ion channels. The subdomain containing Trp-288 is a module tethered to the rest of the extracellular domain by short linkers and intrasubunit interactions between residues in the putative “proton sensor.” Mutations in these two areas shift the apparent affinity of protons toward a more acidic range and change the kinetics of activation and desensitization. These results are consisting with displacement of the module relative to the rest of the extracellular domain to allow interaction of Trp-288 with Tyr-72 during gating. We propose that such interaction may provide functional coupling between the extracellular domain and the pore domain.The acid-sensing ion channels (ASICs)² are voltage-insensitive sodium channels turned on and off by extracellular protons. Four ASIC genes in the human genome, ASIC1 to ASIC4, give rise to at least six isoforms that associate in various combinations to form channels with different functional properties (1, 2). The ASICs constitute a distinct group in the large family of channels known as Degenerins characterized by a common structure but widely diverse gating stimuli: mechanical forces (3), neuropeptides (4, 5), protons (6), or no stimulus at all, such as ENaC, which exhibits constitutive activity (7). The structure shared by all Degenerins consists of two transmembrane segments, TM1 and TM2, a large extracellular domain, and short cytoplasmic amino and carboxyl termini. The recently published crystal structure of a truncated chicken ASIC1 (cASIC1) at a resolution of 1.9 Å (8) shows that ASIC1 is a trimer, and it provides detailed structure of the large extracellular domain that is crucial for understanding the gating mechanism of the ASICs. A feature revealed by the atomic structure is a cluster of negatively and one positively charged residue in the interface of subdomain D (Arg-191), subdomain E (Asp-238 and Glu-239), and subdomain F (Asp-346 and Asp-350) (see Fig. 1A) that was hypothesized to constitute the proton sensor. Furthermore, it was proposed that binding of protons to this site displaces subdomain F toward TM1 to open the pore (8).Open in a separate windowFIGURE 1.Ribbon representation of chicken ASIC1 structure. a, a single subunit is shown for simplicity with subdomains, A to F, indicated in different colors. The arrow points to the putative proton sensor with side chains of charged residues represented as sticks. Amino acids important for ASIC1 gating that were mutated in this study are also shown. The image was obtained with the molecular graphics program Chimera. b, amino acid sequence of subdomain F loop. Residues conserved in all ASIC proteins are in red.Although the solved atomic structure of cASIC1 provides a valuable tool to advance the understanding of how external protons activate the ASICs, it represents only a snapshot of the gating process thereby additional experimental evidence is needed to elucidate the gating mechanism. The general idea that conformational changes triggered by binding of the specific agonist to the extracellular domain of a ligand activated channel need to be transmitted to the transmembrane domain, where the pore gate is located, draws attention to the pair of closely located residues, Tyr-72 and Trp-288, as they provide a potential contact site between the extracellular and the transmembrane domains.This study examines the functional role of the conserved residues, Tyr-72 and Trp-288, that are located distantly in the primary sequence but are brought to close proximity (∼3.7 Å) by the folding of the extracellular domain (ECD). This arrangement could provide a contact site between the ECD and TM1 whereby a conformation change of the ECD is transmitted to the pore gate in the transmembrane domain.     

Fluoroacetic Acid Is a Potent and Specific Inhibitor of Reproduction in the Nematode Caenorhabditis elegans

Fluoroacetic acid is known to lead to inhibition of aconitase and block both the Krebs and glyoxylate cycles. In this study, we discovered it to be a potent and specific inhibitor of reproduction in a bioassay using the nematode Caenorhabditis elegans. Fluoroacetic acid added to the growth medium reduced reproduction in the second generation by 50% at concentrations 3,000 times lower than the concentrations that reduced 24-hour survival by 50%. Four concentrations (2, 4, 8, and 17 mM) of fluoroacetic acid were tested thoroughly. At the two lower concentrations, the survival rates were unaffected, and first-generation reproduction was greatly reduced but not completely eliminated. Survival was reduced at the higher concentrations. Malonate, which inhibits the Krebs cycle, and itaconate, which inhibits the glyoxylate cycle, were tested individually and in combination. The combination did not specifically inhibit reproduction, suggesting another mode of action for fluoroacetic acid. Fluoroacetic acid shows promise as a tool in studies requiring age synchrony.     

Cyclopiazonic Acid Is Complexed to a Divalent Metal Ion When Bound to the Sarcoplasmic Reticulum Ca2+-ATPase

We have determined the structure of the sarco(endo)plasmic reticulum Ca²⁺-ATPase (SERCA) in an E2·P_i-like form stabilized as a complex with , an ATP analog, adenosine 5′-(β,γ-methylene)triphosphate (AMPPCP), and cyclopiazonic acid (CPA). The structure determined at 2.5Å resolution leads to a significantly revised model of CPA binding when compared with earlier reports. It shows that a divalent metal ion is required for CPA binding through coordination of the tetramic acid moiety at a characteristic kink of the M1 helix found in all P-type ATPase structures, which is expected to be part of the cytoplasmic cation access pathway. Our model is consistent with the biochemical data on CPA function and provides new measures in structure-based drug design targeting Ca²⁺-ATPases, e.g. from pathogens. We also present an extended structural basis of ATP modulation pinpointing key residues at or near the ATP binding site. A structural comparison to the Na⁺,K⁺-ATPase reveals that the Phe⁹³ side chain occupies the equivalent binding pocket of the CPA site in SERCA, suggesting an important role of this residue in stabilization of the potassium-occluded E2 state of Na⁺,K⁺-ATPase.The Ca²⁺-ATPase from sarco(endo)plasmic reticulum of rabbit skeletal muscle (SERCA,⁵ isoform 1a) is a thoroughly studied member of the P-type ATPase family (1). SERCA possesses 10 transmembrane helices (M1 through M10) with both the N terminus and the C terminus facing the cytoplasmic side and three cytoplasmic domains, inserted in loops between M2 and M3 (A-domain) and between M4 and M5 (P- and N-domain) (2). The enzyme mediates the uptake of Ca²⁺ ions into the lumen of the sarcoplasmic reticulum (SR) after their release into the cytoplasm through calcium release channels during muscle contraction (3). SERCA, plasma membrane Ca²⁺-ATPase, and a third, Golgi-located secretory pathway Ca²⁺-ATPase are important factors in calcium and manganese homeostasis, transport, signaling, and regulation (4, 5).Crystal structures of all major states in the reaction cycle of SERCA have been determined. These include the Ca₂E₁·ATP state (6, 7) with high affinity Ca²⁺ binding sites accessible from the cytoplasmic side of the SR membrane, the calcium-occluded transition state (6), the open E2P state with luminal facing ion binding sites that have low affinity for Ca²⁺ and high affinity for protons (8) and the proton-occluded H_2–3E2[ATP] state with a bound modulatory ATP (9). This considerable amount of structural information has turned the Ca²⁺-ATPase into a valuable model system for studies on structural rearrangements that take place during the catalytic cycle of P-type ATPases. SERCA is considered a promising drug target in medical research, with a particular focus on prostate cancer and infectious diseases. Several compounds have already been shown to bind and inhibit SERCA by stabilizing the enzyme in a particular conformational state. Thapsigargin (TG), cyclopiazonic acid (CPA), and 2,5-di-(tert-butyl) hydroquinone (BHQ) stabilize an E2-like state, and 1,3-dibromo-2,4,6-tri (methylisothiouronium)benzene stabilizes an E1-P-like conformation (10–13). CPA is a toxic indole tetramic acid first isolated from Penicillium cyclopium (14) and later found to be produced by Aspergillus versicolor and Aspergillus flavus. Like TG, CPA specifically binds to and inhibits SERCA with nanomolar affinity (15). Indeed, CPA is widely used in biochemical and physiological studies on Ca²⁺ signaling and muscle function, where it causes Ca²⁺ store depletion due to specific inhibition of Ca²⁺ reuptake by SERCA. CPA and TG were originally proposed to bind to similar sites on SERCA (16), but recent crystal structures have shown a distinct site of interaction (17, 18). Despite these structural insights, a previously demonstrated magnesium dependence of CPA binding (19) remained unexplained, and opposing CPA binding modes were observed (see below).Tetramic acids are synthesized naturally, and more than 150 natural derivatives have been isolated from bacterial and fungal species (reviewed in Ref. 20). Tetramic acids possessing a 3-acyl group have the ability to chelate divalent metal ions. For instance, tenuazonic acid from the fungus Phoma sorghina has been shown to form complexes with Ca²⁺ and Mg²⁺ (21), as well as heavier metals such as Cu(II), Ni(II), and Fe(III) (22).Previously published crystallographic structures of the SERCA·CPA complex (PDB ID 2O9J and 2EAS) demonstrated that CPA binds within the proposed calcium access channel of SERCA. However, the structures did not reveal a role for magnesium, and the orientation of CPA within this binding site differed in the two studies (17, 18). To address these ambiguities, we have determined the crystal structure of SERCA in complex with , AMPPCP (an ATP analog), and Mn²⁺·CPA. The structure reveals novel insight into CPA binding, which we find to be mediated by a divalent cation, as demonstrated by means of the anomalous scattering properties of Mn²⁺. Further and improved refinement using previously deposited data (PDB ID 2O9J and 2OA0), in light of our new findings, also revealed a strong plausibility for a magnesium ion bound at this site. Furthermore, we find a new configuration of the bound AMPPCP nucleotide, addressing the modulatory role of ATP binding to the E2·P_i occluded conformation of SERCA.     

STK39 Polymorphism Is Associated with Essential Hypertension: A Systematic Review and Meta-Analysis

Background

A recent genome-wide association study identified STK39as a candidate gene for blood pressure (BP) in Europeans. Subsequently, several studies have attempted to replicate the association across different ethnic populations. However, the results have been inconsistent.

Objective and Methods

We performed a meta-analysis to elucidate the association between the STK39 rs3754777 polymorphism (or proxy) and hypertension. Published literature from PubMed and Embase databases were retrieved and pooled odds ratio (OR) with 95% confidence interval (CI) was calculated using fixed- or random-effects model.

Results

Using appropriate inclusion/exclusion criteria, we identified 10 studies that included 21, 863 hypertensive cases and 24, 480 controls from different ethnicities. The meta-analysis showed a significant association of STK39 rs3754777 variant with hypertension (OR = 1.10, 95%CI = 1.06–1.15, p = 7.95×10⁻⁶). Further subgroup analysis by ethnicity suggested that the association was significant in Europeans (OR = 1.08, 95% CI = 1.03–1.14, p = 0.002) and in East Asians (OR = 1.16, 95%CI = 1.07–1.25, p = 4.34×10⁻⁴), but not in Africans (OR = 1.01, 95%CI 0.80–1.27, p = 0.932). We further confirmed the positive association by sensitivity analysis. No publication bias was detected (Begg’s test, p = 0.721; Egger’s test, p = 0.744).

Conclusions

The present meta-analysis confirms the significant association of STK39 polymorphism with susceptibility to hypertension in Europeans and East Asians. Future studies should include gene–gene and gene–environment interactions to investigate the identified association.     

Asymptomatic infection in individuals from the municipality of Barcelos (Brazilian Amazon) is not associated with the anti-Plasmodium falciparum glycosylphosphatidylinositol antibody response

Anti-glycosylphosphatidylinositol (GPI) antibodies (Abs) may reflect and mediate, at least partially, anti-disease immunity in malaria by neutralising the toxic effect of parasitic GPI. Thus, we assessed the anti-GPI Ab response in asymptomatic individuals living in an area of the Brazilian Amazon that has a high level of malaria transmission. For comparative purposes, we also investigated the Ab response to a crude extract prepared from Plasmodium falciparum, the merozoite surface protein (MSP)3 antigen of P. falciparum and the MSP 1 antigen of Plasmodium vivax (PvMSP1-₁₉) in these individuals and in Angolan patients with acute malaria. Our data suggest that the Ab response against P. falciparum GPI is not associated with P. falciparum asymptomatic infection in individuals who have been chronically exposed to malaria in the Brazilian Amazon. However, this Ab response could be related to ongoing parasitaemia (as was previously shown) in the Angolan patients. In addition, our data show that PvMSP1-₁₉may be a good marker antigen to reflect previous exposure to Plasmodium in areas that have a high transmission rate of P. vivax.     

Thyroid Hormone-Regulated Wnt5a/Ror2 Signaling Is Essential for Dedifferentiation of Larval Epithelial Cells into Adult Stem Cells in the Xenopus laevis Intestine

Background and Aims

Amphibian intestinal remodeling, where thyroid hormone (T3) induces some larval epithelial cells to become adult stem cells analogous to the mammalian intestinal ones, serves as a unique model for studying how the adult stem cells are formed. To clarify its molecular mechanisms, we here investigated roles of non-canonical Wnt signaling in the larval-to-adult intestinal remodeling during Xenopus laevis metamorphosis.

Methods/Findings

Our quantitative RT-PCR (qRT-PCR) and immunohistochemical analyses indicated that the expressions of Wnt5a and its receptors, frizzled 2 (Fzd2) and receptor tyrosine kinase-like orphan receptor 2 (Ror2) are up-regulated by T3 and are spatiotemporally correlated with adult epithelial development in the X. laevis intestine. Notably, changes in morphology of larval absorptive epithelial cells expressing Ror2 coincide well with formation of the adult stem cells during metamorphosis. In addition, by using organ cultures of the tadpole intestine, we have experimentally shown that addition of exogenous Wnt5a protein to the culture medium causes morphological changes in the larval epithelium expressing Ror2 even in the absence of T3. In contrast, in the presence of T3 where the adult stem cells are formed in vitro, inhibition of endogenous Wnt5a by an anti-Wnt5a antibody suppressed the epithelial morphological changes, leading to the failure of stem cell formation.

Significance

Our findings strongly suggest that the adult stem cells originate from the larval absorptive cells expressing Ror2, which require Wnt5a/Ror2 signaling for their dedifferentiation accompanied by changes in cell morphology.     

dRecQ4 Is Required for DNA Synthesis and Essential for Cell Proliferation in Drosophila

Background

The family of RecQ DNA helicases plays an important role in the maintenance of genomic integrity. Mutations in three of the five known RecQ family members in humans, BLM, WRN and RecQ4, lead to disorders that are characterized by predisposition to cancer and premature aging.

Methodology/Principal Findings

To address the in vivo functions of Drosophila RecQ4 (dRecQ4), we generated mutant alleles of dRecQ4 using the targeted gene knock-out technique. Our data show that dRecQ4 mutants are homozygous lethal with defects in DNA replication, cell cycle progression and cell proliferation. Two sets of experiments suggest that dRecQ4 also plays a role in DNA double strand break repair. First, mutant animals exhibit sensitivity to gamma irradiation. Second, the efficiency of DsRed reconstitution via single strand annealing repair is significantly reduced in the dRecQ4 mutant animals. Rescue experiments further show that both the N-terminal domain and the helicase domain are essential to dRecQ4 function in vivo. The N-terminal domain is sufficient for the DNA repair function of dRecQ4.

Conclusions/Significance

Together, our results show that dRecQ4 is an essential gene that plays an important role in not only DNA replication but also DNA repair and cell cycle progression in vivo.     

Systematics of the Osteocephalus buckleyi species complex (Anura,Hylidae) from Ecuador and Peru

We present a new phylogeny, based on DNA sequences of mitochondrial and nuclear genes, for frogs of the genus Osteocephalus with emphasis in the Osteocephalus buckleyi species complex. Genetic, morphologic, and advertisement call data are combined to define species boundaries and describe new species. The phylogeny shows strong support for: (1) a basal position of Osteocephalus taurinus + Osteocephalus oophagus, (2) a clade containing phytotelmata breeding species, and (3) a clade that corresponds to the Osteocephalus buckleyi species complex. Our results document a large proportion of hidden diversity within a set of populations that were previously treated as a single, widely distributed species, Osteocephalus buckleyi. Individuals assignable to Osteocephalus buckleyi formed a paraphyletic group relative to Osteocephalus verruciger and Osteocephalus cabrerai and contained four species, one of which is Osteocephalus buckleyi sensu stricto and three are new. Two of the new species are shared between Ecuador and Peru (Osteocephalus vilmae sp. n. and Osteocephalus cannatellai sp. n.) and one is distributed in the Amazon region of southern Peru (Osteocephalus germani sp. n.) We discuss the difficulties of using morphological characters to define species boundaries and propose a hypothesis to explain them.     

3-Methylcrotonyl-Coenzyme A Carboxylase Is a Component of the Mitochondrial Leucine Catabolic Pathway in Plants

3-Methylcrotonyl-coenzyme A carboxylase (MCCase) is a mitochondrial biotin-containing enzyme whose metabolic function is not well understood in plants. In soybean (Glycine max) seedlings the organ-specific and developmentally induced changes in MCCase expression are regulated by mechanisms that control the accumulation of MCCase mRNA and the activity of the enzyme. During soybean cotyledon development, when seed-storage proteins are degraded, leucine (Leu) accumulation peaks transiently at 8 d after planting. The coincidence between peak MCCase expression and the decline in Leu content provides correlative evidence that MCCase is involved in the mitochondrial catabolism of Leu. Direct evidence for this conclusion was obtained from radiotracer metabolic studies using extracts from isolated mitochondria. These experiments traced the metabolic fate of [U-¹⁴C]Leu and NaH¹⁴CO₃, the latter of which was incorporated into methylglutaconyl-coenzyme A (CoA) via MCCase. These studies directly demonstrate that plant mitochondria can catabolize Leu via the following scheme: Leu → α-ketoisocaproate → isovaleryl-CoA → 3-methylcrotonyl-CoA → 3-methylglutaconyl-CoA → 3-hydroxy-3-methylglutaryl-CoA → acetoacetate + acetyl-CoA. These findings demonstrate for the first time, to our knowledge, that the enzymes responsible for Leu catabolism are present in plant mitochondria. We conclude that a primary metabolic role of MCCase in plants is the catabolism of Leu.     

Key for European species of the Cheilosia proxima group (Diptera,Syrphidae) with a description of a new species

A new hoverfly species, Cheilosia barbafacies Vujić & Radenković sp. n. (Diptera, Syrphidae), is described and distinguished from the closely related species Cheilosia pascuorum Becker, 1894, based on material collected from the mountains of the Balkan Peninsula. Diagnostic characteristics and an identification key for the members of the proxima group of Cheilosia s. str., including the new taxon, are provided.     

Changes in Ucp1, D2 (Dio2) and Glut4 (Slc2a4) mRNA expression in response to short-term cold exposure in the house musk shrew (Suncus murinus).

The house musk shrew (Suncus murinus), or so-called suncus, is a cold-intolerant mammal, but it is unclear why it is susceptible to low temperatures. Cold-intolerance may be the result of lower thermogenic activity in brown adipose tissue (BAT). The early phase of severe cold exposure is a critical period for suncus. Therefore, we exposed suncus to mildly cold temperatures (10-12 degrees C) for 1 to 48 h to increase non-shivering thermogenesis without causing stress and measured changes in the expression of uncoupling protein 1 (Ucp1), type II iodothyronine 5'-deiodinase (Dio2=D2), and glucose transporter 4 (Slc2a4=Glut4) in BAT. These mRNAs play a major role in non-shivering thermogenesis and are mainly regulated by the sympathetic nervous system via direct beta-noradrenergic innervation of BAT. During cold exposure, Ucp1 expression in BAT increased steadily over time, albeit only slightly. Neither D2 nor Glut4 expression in BAT increased immediately; however, they had increased significantly after 24 h and 48 h of cold exposure. These findings suggest that the responsiveness of mRNA regulation is weak and thus may be involved in cold-intolerance in suncus.     

The 5 kDa Protein NdhP Is Essential for Stable NDH-1L Assembly in Thermosynechococcus elongatus

The cyanobacterial NADPH:plastoquinone oxidoreductase complex (NDH-1), that is related to Complex I of eubacteria and mitochondria, plays a pivotal role in respiration as well as in cyclic electron transfer (CET) around PSI and is involved in a unique carbon concentration mechanism (CCM). Despite many achievements in the past, the complex protein composition and the specific function of many subunits of the different NDH-1 species remain elusive. We have recently discovered in a NDH-1 preparation from Thermosynechococcus elongatus two novel single transmembrane peptides (NdhP, NdhQ) with molecular weights below 5 kDa. Here we show that NdhP is a unique component of the ∼450 kDa NDH-1L complex, that is involved in respiration and CET at high CO₂ concentration, and not detectable in the NDH-1MS and NDH-1MS'' complexes that play a role in carbon concentration. C-terminal fusion of NdhP with his-tagged superfolder GFP and the subsequent analysis of the purified complex by electron microscopy and single particle averaging revealed its localization in the NDH-1L specific distal unit of the NDH-1 complex, that is formed by the subunits NdhD1 and NdhF1. Moreover, NdhP is essential for NDH-1L formation, as this type of NDH-1 was not detectable in a ΔndhP::Km mutant.     

On the Monacha species of Lebanon (Gastropoda,Hygromiidae)

In this paper, all seven hitherto known species of the hygromiid genus Monacha from Lebanon are briefly characterised and illustrated, and distribution maps are supplied (Monacha (Monacha) syriaca (Ehrenberg, 1831), Monacha (Monacha) nummus (Ehrenberg, 1831), Monacha (Monacha) obstructa (L. Pfeiffer, 1842), Monacha (Monacha) crispulata (Mousson, 1861), Monacha (Monacha) solitudinis (Bourguignat, 1852), Monacha (Monacha) bari Forcart 1981, and Monacha (Monacha) cf. compingtae (Pallary, 1929)). One species, Monacha (Monacha) bari Forcart, 1981, is recorded for the country for the first time, and its relationship to Monacha (Monacha) compingtae (Pallary, 1929) is discussed. Based on recently collected specimens, the genital organs of a long time ignored species, Helix solitudinis Bourguignat, 1852 could be investigated. It is here re-described as a Monacha species endemic for Lebanon.