Immunological properties of notexin, a potent presynaptic and myotoxic component from venom of the Australian tiger snake Notechis scutatus scutatus

Neutralizing antibodies were raised in mice against notexin, the most toxic phospholipase A₂ (PLA₂) from Notechis scutatus scutatus venom, without the necessity of detoxifying the toxin prior to immunization. Using a sensitive radioimmunoassay we demonstrated that anti-notexin antibodies recognized (i) the parent antigen, (ii) closely related isoforms of notexin and (iii) venoms from Notechis genus snakes. In contrast, they failed to recognize other purified PLA₂ or PLA₂-containing venoms from other origins. Substitutions or chemical modifications occurring in the C-terminal part of the polypeptide chain of notexin altered the binding affinity for antibodies, implying that this region constitutes an antigenic domain of notexin.     

Ergosterol peroxides as phospholipase A2 inhibitors from the fungus Lactarius hatsudake

Four ergosterol derivatives (1–4) have been isolated for the first time from the fruiting bodies of a basidiomycete fungus, Lactarius hatsudake, through activity-guided fractionation. Their structures were determined, using spectroscopic analysis, as: (22E,24R)-ergosta-5,7,22-dien-3β-ol (ergosterol, 1); 5,8-epidioxy-(22E,24R)-ergosta-6,22-dien-3β-ol (ergosterol peroxide, 2); 5,8-epidioxy-(24S)-ergosta-6-en-3β-ol (3); and (22E,24R)-ergosta-7,22-dien-3β,5,6β-triol (cerevisterol, 4). Compounds 2 and 3 showed selective inhibitory activity against Crotalus adamenteus venom phospholipase A₂ (PLA₂) enzyme, but not against Apis mellifcra bee venom PLA₂. The antiphospholipase A₂ activity of compounds 2 and 3 are reported here for the first time.     

Snake venomics and antivenomics of the arboreal neotropical pitvipers Bothriechis lateralis and Bothriechis schlegelii

We report the comparative proteomic characterization of the venoms of two related neotropical arboreal pitvipers from Costa Rica of the genus Bothriechis, B. lateralis (side-striped palm pit viper) and B. schlegelii (eyelash pit viper). The crude venoms were fractionated by reverse-phase HPLC, followed by analysis of each chromatographic fraction by SDS-PAGE, N-terminal sequencing, MALDI-TOF mass fingerprinting, and collision-induced dissociation tandem mass spectrometry of tryptic peptides. The venom proteomes of B. lateralis and B. schlegelii comprise similar number of distinct proteins belonging, respectively, to 8 and 7 protein families. The two Bothriechis venoms contain bradykinin-potentiating peptides (BPPs), and proteins from the phospholipase A 2 (PLA 2), serine proteinase, l-amino acid oxidase (LAO), cysteine-rich secretory protein (CRISP), and Zn (2+)-dependent metalloproteinase (SVMP) families, albeit each species exhibit different relative abundances. Each venom also contains unique components, for example, snake venom vascular endothelial growth factor (svVEGF) and C-type lectin-like molecules in B. lateralis, and Kazal-type serine proteinase inhibitor-like proteins in B. schlegelii. Using a similarity coefficient, we estimate that the similarity of the venom proteins between the two Bothriechis taxa may be <10%, indicating a high divergence in their venom compositions, in spite of the fact that both species have evolved to adapt to arboreal habits. The major toxin families of B. lateralis and B. schlegelii are SVMP (55% of the total venom proteins) and PLA 2 (44%), respectively. Their different venom toxin compositions provide clues for rationalizing the distinct signs of envenomation caused by B. schlegelii and B. lateralis. An antivenomic study of the immunoreactivity of the Instituto Clodomiro Picado (ICP) polyvalent antivenom toward Bothriechis venoms revealed that l-amino acid oxidase and SVMPs represent the major antigenic protein species in both venoms. Our results provide a ground for rationalizing the reported protection of the ICP polyvalent antivenom against the hemorrhagic, coagulant, defibrinating, caseinolytic and fibrin(ogen)olytic activities of Bothriechis ( schlegelii, lateralis) venoms. However, these analyses also evidenced the limited recognition capability of the polyvalent antivenom toward a number of Bothriechis venom components, predominantly BPPs, svVEGF, Kazal-type inhibitors, some PLA 2 proteins, some serine proteinases, and CRISP molecules.     

Properties of the Group IV phospholipase A2 family

The Group IV phospholipase A₂ family is comprised of six intracellular enzymes commonly called cytosolic phospholipase A₂ (cPLA₂) , cPLA₂β, cPLA₂γ, cPLA₂δ, cPLA₂ε and cPLA₂ζ. They are most homologous to phospholipase A and phospholipase B/lysophospholipases of filamentous fungi particularly in regions containing conserved residues involved in catalysis. However, a number of other serine acylhydrolases (patatin, Group VI PLA₂s, Pseudomonas aeruginosa ExoU and NTE) contain the Ser/Asp catalytic dyad characteristic of Group IV PLA₂s, and recent structural analysis of patatin has confirmed its structural similarity to cPLA₂. A characteristic of all these serine acylhydrolases is their ability to carry out multiple reactions to varying degrees (PLA₂, PLA₁, lysophospholipase and transacylase activities). cPLA₂, the most extensively studied Group IV PLA₂, is widely expressed in mammalian cells and mediates the production of functionally diverse lipid products in response to extracellular stimuli. It has PLA₂ and lysophospholipase activities and is the only PLA₂ that has specificity for phospholipid substrates containing arachidonic acid. Because of its role in initiating agonist-induced release of arachidonic acid for the production of eicosanoids, cPLA₂ activation is important in regulating normal and pathological processes in a variety of tissues. Current information available about the biochemical properties and tissue distribution of other Group IV PLA₂s suggests they may have distinct mechanisms of regulation and functional roles.     

Determination of the maximum and minimum lethal temperatures (LT50) for Loxosceles intermedia Mello-Leitão, 1934 and L. laeta (Nicolet, 1849) (Araneae, Sicariidae)

In this study, the maximum and minimum lethal temperatures (LT₅₀) of L. intermedia and L. laeta were determined in two treatments: gradual heating (25–50°C) and cooling (25°C to −5°C), and 1 h at a constant temperature. In gradual temperatures change, L. intermedia mortality started at 40°C and the LT₅₀ was 42°C; for L. laeta, mortality began at 35°C and the LT₅₀ was 40°C. At low temperatures, mortality was registered only at −5°C for both species. In the constant temperature L. intermedia showed a maximum LT₅₀ at 35°C and L. laeta at 32°C; the minimum LT for both species was −7°C.     

Venom peptide analysis of Vipera ammodytes meridionalis (Viperinae) and Bothrops jararacussu (Crotalinae) demonstrates subfamily-specificity of the peptidome in the family Viperidae

Snake venom peptidomes are valuable sources of pharmacologically active compounds. We analyzed the peptidic fractions (peptides with molecular masses < 10,000 Da) of venoms of Vipera ammodytes meridionalis (Viperinae), the most toxic snake in Europe, and Bothrops jararacussu (Crotalinae), an extremely poisonous snake of South America. Liquid chromatography/mass spectrometry (LC/MS), direct infusion electrospray mass spectrometry (ESI-MS) and matrix-assisted desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) were applied to characterize the peptides of both snake venoms. 32 bradykinin-potentiating peptides (BPPs) were identified in the Crotalinae venom and their sequences determined. 3 metalloproteinase inhibitors, 10 BPPs and a Kunitz-type inhibitor were observed in the Viperinae venom peptidome. Variability in the C-terminus of homologous BPPs was observed, which can influence the pharmacological effects. The data obtained so far show a subfamily specificity of the venom peptidome in the Viperidae family: BPPs are the major peptide component of the Crotalinae venom peptidome lacking Kunitz-type inhibitors (with one exception) while the Viperinae venom, in addition to BPPs, can contain peptides of the bovine pancreatic trypsin inhibitor family. We found indications for a post-translational phosphorylation of serine residues in Bothrops jararacussu venom BPP (S[combining low line]QGLPPGPPIP), which could be a regulatory mechanism in their interactions with ACE, and might influence the hypotensive effect. Homology between venom BPPs from Viperidae snakes and venom natriuretic peptide precursors from Elapidae snakes suggests a structural similarity between the respective peptides from the peptidomes of both snake families. The results demonstrate that the venoms of both snakes are rich sources of peptides influencing important physiological systems such as blood pressure regulation and hemostasis. The data can be used for pharmacological and medical applications.     

Production of phosphatidylcholine containing conjugated linoleic acid mediated by phospholipase A2

Esterification of lysophosphatidylcholine (LPC) with conjugated linoleic acid (CLA) was carried out using porcine pancreatic phospholipase A₂ (PLA₂). PLA₂ only slightly synthesized phosphatidylcholine containing CLA (CLA-PC) at 2.6% by the addition of water. Addition of formamide in place of water markedly increased the yield of CLA-PC. In addition, synthesis of CLA-PC by PLA₂ was affected by the amount of substrate CLA and PLA₂ in the reaction system. Under optimal reaction conditions using 11 mg LPC, 18 mg CLA, 550 mg glycerol, 50 μL formamide, 3.3 × 10⁴ U PLA₂, and 0.3 μmol CaCl₂ at 37 °C for 6 h, the reaction yield of CLA-PC reached 65 mol%. Furthermore, addition of protein such as albumin and casein suppressed the decrease of CLA-PC yield after 6 h. PLA₂ exhibited the highest activity for the 10t,12c-CLA isomer among four CLA isomers (9c,11t-CLA, 9c,11c-CLA, 9t,11t-CLA and 10t,12c-CLA), whereas that for 9c,11c-CLA was the lowest. These results showed that the present esterification system for LPC and CLA by PLA₂ is effective for producing CLA-PC.     

Metal–salt co-tolerance and metal removal by indigenous cyanobacterial strains

Chromium and salt tolerance in five indigenous cyanobacterial strains isolated from contaminated sites was investigated along with their metal bioaccumulative potential. All the five species showed significantly better growth when the medium was spiked with salt or chromium. As compared to single metal or salt treatment, the binary metal–salt (MS) treatments had more favorable effect on cyanobacterial growth as indicated by significantly higher concentration of the primary photosynthetic pigment chlorophyll at M₂₀S₂₀₀₀ (9.9–25.3 μg/mL) as compared to that at M₀S₀ (4.0–12.3 μg/mL). Similarly biomass was much higher at M₂₀S₁₀₀₀ and M₂₀S₂₀₀₀ (41.8–86.2 mg/10 mL) as compared to that at control, M₀S₀ (21.5–36.3 mg/10 mL). Accessory pigments like carotenoids and phycobilinproteins too tended to increase significantly in response to both metal and salts in the two species of Lyngbya (L. putealis and L. ceylanica var. constricta) and Gloeocapsa. These species also showed greater potential of chromium bioaccumulation, which increased further as both salt and metal concentration increased. In the two species of Nostoc however, bioaccumulative potential improve at higher metal concentration, but not affected significantly by salt concentration.     

Venomic analysis and evaluation of antivenom cross-reactivity of South American Micrurus species

Coral snakes from Micrurus genus are the main representatives of the Elapidae family in South America. However, biochemical and pharmacological features regarding their venom constituents remain poorly investigated. Here, venomic analyses were carried out aiming at a deeper understanding on the composition of M. frontalis, M. ibiboboca, and M. lemniscatus venoms. In the three venoms investigated, proteins ranging from 6 to 8 kDa (3FTx) and 12 to 14 kDa (PLA(2)) were found to be the most abundant. Also, the N-terminal sequences of four new proteins, purified from the M. lemniscatus venom, similar to 3FTx, PLA(2) and Kunitz-type protease inhibitor from other Micrurus and elapid venoms are reported. Cross-reactivity among different Micrurus venoms and homologous or heterologous antivenoms was carried out by means of 2D-electrophoresis and immunoblotting. As, expected, the heterologous anti-Elapid venom displayed the highest degree of cross-reactivity. Conversely, anti-M. corallinus reacted weakly against the tested venoms. In gel digestions, followed by mass spectrometry sequencing and similarity searching, revealed the most immunogenic protein families as similar to short and long neurotoxins, weak neurotoxins, PLA(2), β-bungarotoxin, venom protein E2, frontoxin III, LAO and C-type lectin. The implications of our results for the production of Micrurus antivenoms are discussed.     

Primary structure and biological activity of bradykinin potentiating peptides fromBothrops insularis snake venom

Several bradykinin potentiating peptides (BPPs) were isolated from the venom of the Brazilian arboricole snake Bothrops insularis by gel filtration on Sephadex G-150-120, followed by sequencial high-voltage paper electrophoreses atpH 3.5, 6.5, and 2.1. The BPPs were assayed by their ability to potentiate the contractile activity, on the isolated guinea pig ileum, and the hypotensive activity, on anesthetized rats, of bradykinin. Eight BPPs, containing 3–13 amino acid residues, were sequenced and their primary structures were shown to have a marked degree of homology with those of several BPPs from other venoms.     

A novel peptide from the ACEI/BPP-CNP precursor in the venom of Crotalus durissus collilineatus

In crotaline venoms, angiotensin-converting enzyme inhibitors [ACEIs, also known as bradykinin potentiating peptides (BPPs)], are products of a gene coding for an ACEI/BPP-C-type natriuretic peptide (CNP) precursor. In the genes from Bothrops jararaca and Gloydius blomhoffii, ACEI/BPP sequences are repeated. Sequencing of a cDNA clone from venom glands of Crotalus durissus collilineatus showed that two ACEIs/BPPs are located together at the N-terminus, but without repeats. An additional sequence for CNP was unexpectedly found at the C-terminus. Homologous genes for the ACEI/BPP-CNP precursor suggest that most crotaline venoms contain both ACEIs/BPPs and CNP. The sequence of ACEIs/BPPs is separated from the CNP sequence by a long spacer sequence. Previously, there was no evidence that this spacer actually coded any expressed peptides. Aird and Kaiser (1986, unpublished) previously isolated and sequenced a peptide of 11 residues (TPPAGPDVGPR) from Crotalus viridis viridis venom. In the present study, analysis of the cDNA clone from C. d. collilineatus revealed a nearly identical sequence in the ACEI/BPP-CNP spacer. Fractionation of the crude venom by reverse phase HPLC (C(18)), and analysis of the fractions by mass spectrometry (MS) indicated a component of 1020.5 Da. Amino acid sequencing by MS/MS confirmed that C. d. collilineatus venom contains the peptide TPPAGPDGGPR. Its high proline content and paired proline residues are typical of venom hypotensive peptides, although it lacks the usual N-terminal pyroglutamate. It has no demonstrable hypotensive activity when injected intravenously in rats; however, its occurrence in the venoms of dissimilar species suggests that its presence is not accidental. Evidence suggests that these novel toxins probably activate anaphylatoxin C3a receptors.     

Lactobacillus harbinensis sp. nov., consisted of strains isolated from traditional fermented vegetables 'Suan cai' in Harbin, Northeastern China and Lactobacillus perolens DSM 12745

Taxonomical analysis of two genetically distinguished Lactobacillus strains isolated from traditional Chinese fermented vegetables ‘Suan cai’ was performed. They formed l-lactate from glucose, were facultatively heterofermentative, and had a DNA G+C content of 53–54 mol%. They fermented d- and l-arabinose. They produced lactate, ethanol and acetate from gluconate at a molar ratio of 1.1:0.4:0.7. Phylogenetic analysis of 16S rDNA revealed that the two strains were closely related to L. perolens. DNA–DNA hybridization analysis revealed that the two strains were different from L. perolens type strain DSM 12744 and formed a separate cluster with L. perolens DSM 12745. G+C molar content of DNA of the former is 51%, whereas those of the latter strains were in the range of 53–54%. Based on the results, we propose that the new species be named L. harbinensis sp. nov. and that L. perolens DSM 12745 be reclassified as L. harbinensis DSM 12745. The type strain of L. harbinensis DSM 16991^T (=AHU 1762^T=SBT 10908^T).     

Effect of leaf age on CAM activity in Littorella uniflora

In this study the effect of ontogenetic drift on crassulacean acid metabolism (CAM) was investigated in the aquatic CAM-isoetid Littorella uniflora. The results of this study strengthen the general hypothesis of CAM being a carbon-conserving mechanism in aquatic plants, because high-CAM capacity (45–183 μequiv. g⁻¹ FW) was present in all leaves of L. uniflora irrespective of age. Since possession of CAM in aquatic plants allows CO₂ uptake throughout the light/dark cycle, presence of CAM in all leaves influences the carbon balance of L. uniflora positively. On average for all lakes, different leaf classes accounted for 11–36% of the total dark CO₂ uptake by the individual plant.

The capacity for both CAM and photosynthesis declined with increasing leaf age, and was in the oldest leaves only 25–53% of the capacity in the youngest. The photosynthetic capacity was estimated to be sufficiently high to ensure refixation of the CO₂ released from malate during decarboxylation in the daytime. In line with this, a linear coupling between CAM capacity and photosynthetic capacity was found. Parallel to the change in photosynthetic capacity, an age-related change in total ribulose-bisphosphate carboxylase/oxygenase (rubisco) activity from 732 μmol C g⁻¹ DW h⁻¹ in the youngest leaves to 346 μmol C g⁻¹ DW h⁻¹ in the oldest was observed. In contrast, no significant change in phosphoenolpyruvate carboxylase (PEPcase) activity with leaf age was observed (means ranged between 46 and 156 μmol C g⁻¹ DW h⁻¹).     

A multifaceted analysis of viperid snake venoms by two-dimensional gel electrophoresis: an approach to understanding venom proteomics

The complexity of Viperid venoms has long been appreciated by investigators in the fields of toxinology and medicine. However, it is only recently that the depth of that complexity has become somewhat quantitatively and qualitatively appreciated. With the resurgence of two-dimensional gel electrophoresis (2-DE) and the advances in mass spectrometry virtually all venom components can be visualized and identified given sufficient effort and resources. Here we present the use of 2-DE for examining venom complexity as well as demonstrating interesting approaches to selectively delineate subpopulations of venom proteins based on particular characteristics of the proteins such as antibody cross-reactivity or enzymatic activities. 2-DE comparisons between venoms from different species of the same genus (Bothrops) of snake clearly demonstrated both the similarity as well as the apparent diversity among these venoms. Using liquid chromatography/tandem mass spectrometry we were able to identify regions of the two-dimensional gels from each venom in which certain classes of proteins were found. 2-DE was also used to compare venoms from Crotalus atrox and Bothrops jararaca. For these venoms a variety of staining/detection protocols was utilized to compare and contrast the venoms. Specifically, we used various stains to visualize subpopulations of the venom proteomes of these snakes, including Coomassie, Silver, Sypro Ruby and Pro-Q-Emerald. Using specific antibodies in Western blot analyses of 2-DE of the venoms we have examined subpopulations of proteins in these venoms including the serine proteinase proteome, the metalloproteinase proteome, and the phospholipases A2 proteome. A functional assessment of the gelatinolytic activity of these venoms was also performed by zymography. These approaches have given rise to a more thorough understanding of venom complexity and the toxins comprising these venoms and provide insights to investigators who wish to focus on these venom subpopulations of proteins in future studies.     

Orpotrin: a novel vasoconstrictor peptide from the venom of the Brazilian stingray Potamotrygon gr. orbignyi

Characterization of the peptide content of venoms has a number of potential benefits for basic research, clinical diagnosis, development of new therapeutic agents, and production of antiserum. In order to analyze in detail the peptides and small proteins of crude samples, techniques such as chromatography and mass spectrometry have been employed. The present study describes the isolation, biochemical characterization, and sequence determination of a novel peptide, named Orpotrin from the venom of Potamotrygon gr. orbignyi. The natural peptide was shown to be effective in microcirculatory environment causing a strong vasoconstriction. The peptide was fully sequenced by de novo amino acid sequencing with mass spectrometry and identified as the novel peptide. Its amino acid sequence, HGGYKPTDK, aligns only with creatine kinase residues 97–105, but has no similarity to any bioactive peptide. Therefore, possible production of this peptide from creatine kinase by limited proteolysis is discussed. Taken together, the results indicate the usefulness of this single-step approach for low molecular mass compounds in complex samples such as venoms.     

In vitro antiprotozoal activity of the lipophilic extracts of different parts of Turkish Pistacia vera L.

Thirteen lipophilic extracts prepared with n-hexane from various parts of Pistacia vera L. tree (Anacardiaceae) growing in Turkey were screened for their in vitro activity against four parasitic protozoa, Trypanosoma brucei rhodesiense, Trypanosoma cruzi, Leishmania donovani and Plasmodium falciparum. Melarsoprol, benznidazole, miltefosine, artemisinin and chloroquine were used as reference drugs. The cytotoxic potentials of the extracts on rat skeletal myoblast (L6) cells were also assessed and compared to that of podophyllotoxin. The screening method employed was medium-throughput, where the extracts were tested at two concentrations, at 0.8 and 4.8 μg/ml (T. brucei rhodesiense, L. donovani and Plasmodium falciparum), or at 1.6 and 9.7 μg/ml (T. cruzi and L6 cells). At 4.8 μg/ml concentration, the branch extract of Pistacia vera (PV-BR) significantly inhibited (77.3%) the growth of L. donovani, whereas the dry leaf extract (PV-DL) was active against Plasmodium falciparum (60.6% inhibition). The IC₅₀ values of these extracts were determined as 2.3 μg/ml (PV-BR, L. donovani) and 3.65 μg/ml (PV-DL, Plasmodium falciparum). None of the extracts possessed cytotoxicity on mammalian cells.     

The effect of shielding the parietal eye of Podarcis muralis on behavioral thermoregulation

1. 1. Body temperatures (T_bs) and thermoregulatory precision of 7 sham shielded and 7 parietal eye shielded Podarcis muralis were measured in a linear thigmothermal gradient over a 24 h period.

2. 2. Shielding the parietal eye did not alter the mean T_b selected over the 24 h period.

3. 3. Both groups selected T_bs that did not differ between photophase and scotophase.

4. 4. Shielding the parietal eye did not influence thermoregulatory precision when measured over the 12 h of photophase, but from 0600–1200 h EST parietal eye shielded lizards thermoregulated more precisely than sham shielded lizards.

     

A trap for in situ cultivation of filamentous actinobacteria

The number of pathogens involved in community-acquired pneumonia, with varying susceptibilities to antimicrobials, is numerous constituting an enormous challenge for diagnostic microbiology. Differentiation of infections due to Streptococcus pneumoniae and those due to Mycoplasma pneumoniae, Chlamydophila pneumoniae, or L. pneumophila as well as those due to viruses is essential to allow correct decisions concerning the antibiotics to be administered.

The sensitivity and specificity of real-time simplex and multiplex nucleic acid sequence-based amplification (NASBA), and simplex PCR were compared for the detection of M. pneumoniae, C. pneumoniae and Legionella spp. in respiratory specimens from hospitalized and outpatients with community-acquired pneumonia (CAP).

Two hundred fifty one respiratory specimens were collected from 147 patients with CAP. NASBA was done using the NucliSens Basic Kit (bioMérieux). PCR for M. pneumoniae and C. pneumoniae was done as described earlier [Ieven, M., Ursi, D., Van Bever, H., Quint, W., Niesters, H. G. M., and Goossens, H. 1996. Detection of Mycoplasma pneumoniae by two polymerase chain reactions and role of M. pneumoniae in acute respiratory tract infections in pediatric patients. J. Infect. Dis. 173, 1445–14452.; Ursi, D., Ieven, M., Van Bever, H. P., and Goossens, H. 1998. Construction of an internal control for the detection of Chlamydia pneumoniae by PCR. Mol. Cellul. Probes. 12, 235–238.]. A real-time PCR was developed to detect L. pneumophila whereas a real-time NASBA was designed to detect Legionella spp. All samples with discordant results were re-analysed.

Compared to an expanded gold standard the sensitivities of the different techniques, were 77.8%, 100%, and 100% for detection of M. pneumoniae; and 50%, 100%, and 50% for detection of L. pneumophila by PCR, real-time simplex NASBA, and real-time multiplex NASBA, respectively.

C. pneumoniae was detected in two samples only.

Simplex real-time NASBA proved to be more sensitive than simplex PCR and was also more sensitive than real-time multiplex NASBA, as previously found with spiked clinical specimens. It's practical attractiveness pleads for further optimalisation of the multiplex approach.     

Comparative analysis of structure, expression and PSD95-binding capacity of Lrfn, a novel family of neuronal transmembrane proteins

Leucine-rich repeat and fibronectin III domain-containing (Lrfn) has five members in mouse and human (Lrfn1, Lrfn2, Lrfn3, Lrfn4, Lrfn5), and homologues in other vertebrates. Lrfn proteins share leucine-rich repeat (LRR)–immunoglobulin-like (Ig)–fibronectin type III (Fn)–transmembrane domain structure, which is also found in LRR–Ig–Fn superfamily proteins. Mouse Lrfn genes were expressed at adult stage predominantly in the brain. In the course of development, expression of Lrfn1, Lrfn3, and Lrfn4 started from immature neural cells, whereas that of Lrfn2 and Lrfn5 was limited to mature ones. Lrfn1–5 commonly encode glycoproteins spanning the plasma membrane, with their N-terminus located on the extracellular side. C-termini of Lrfn1, Lrfn2 and Lrfn4 were bound by PDZ domains of postsynaptic protein PSD95, re-distributing PSD95 to cell periphery where the Lrfn proteins were detected. These results suggest that Lrfn proteins are neuronal components with a role in the developing or mature vertebrate nervous system.     

Sequence analysis of mouse cDNAs encoding ribosomal proteins L12 and L18

Mouse cDNAs encoding ribosomal proteins (r-proteins), L12 and L18, were isolated and their sequences determined. The L12 cDNA was found to contain 639 bp, including a coding sequence of 498 nucleotides (nt), 5' (78 nt) and 3' (45 nt) untranslated regions (UTRs), and a poly(A) tail of 18 nt. The L18 cDNA was shown to consist of 648 bp, including a coding sequence of 567 nt, 5' (26 nt) and 3' (39 nt) UTRs, and a poly(A) tail of 16 nt. The nt sequences of the protein-coding region from the mouse L12 and L18 cDNAs were found to exhibit 96% and 92% identity, respectively, with those of the rat. With the use of mouse L12 and L18 cDNA probes, multiple (at least 10) copies of the L12 and L18 gene families were shown to be present in the mouse and rat genomes. However, there was no sequence heterogeneity detected among seven L18 cDNA clones, indicating that only one copy of the L18 gene-related sequences is functional, and the other copies are presumably nonfunctional pseudogenes. The complete amino acid (aa) sequences of the mouse r-proteins, L12 and L18, were deduced from the nt sequences of their cDNA clones. L12 has 165 aa and a M_r, of 17 790, while L18 has 188 aa and a M_r of 21 570. The aa sequences of the mouse r-proteins, L12 and L18, exhibit 98% and 94% identity, respectively, to those of rat.