Assessing the in vivo toxicity of titanium dioxide nanoparticles in Schmidtea mediterranea: uptake pathways and (neuro)developmental outcomes.

Titanium dioxide nanoparticles (TiO2-NPs) in aquatic environments, originating from urban run-off, product use and post-consumer degradation, interact with aquatic organisms through water and sediments. Thorough toxicity assessment requires comprehensive data across all ecosystem compartments especially the benthic zone, which is currently lacking. Moreover, a proper physicochemical characterization of the particles is needed before and during toxicity assessment. In the present work, we used the planarian Schmidtea mediterranea to investigate the effects of TiO2-NPs (5 mg/L and 50 mg/L). Planarians are benthic organisms that play an important role in the food chain as predators. Our study integrated particle characterization with toxicokinetic and toxicodynamic parameters and showed that the uptake of TiO2-NPs of 21 nm occurred through the epidermis and intestine. Epidermal irritation and mucus production occurred immediately after exposure, and TiO2-NPs induced stronger effects in regenerating organisms. More specifically, TiO2-NPs interfered with neuroregeneration, inducing behavioral effects. A delay in the formation of the anterior commissure between the two brain lobes after seven and nine days of exposure to 50 mg/L was observed, probably as a result of a decrease in stem cell proliferation. Our findings underscore the need to incorporate multiple exposure routes in toxicity screenings. Additionally, we highlight the vulnerability of developing organisms and recommend their inclusion in future risk assessment strategies.

Changes in coronal knee-alignment parameters during the osteoarthritis process in the varus knee.

OBJECTIVES: The idea to aim for an "individualized" alignment, whereby the constitutional alignment is restored, has gained much interest among knee surgeons. This requires insight into the prediseased, natural alignment of our patients' knees. The aim of this study is (1) to determine how the hip-knee-ankle (HKA) angle is influenced during the arthritic process and (2) to investigate the correlation between joint line changes and the progression of osteoarthritis (OA). It is our hypothesis that the most pronounced coronal parameter changes appear at the proximal tibia and at the joint line. METHODS: One hundred sequential full-length X-rays with a minimum follow-up of 1 year were retrospectively reviewed from a radiographic joint database. Patients had to be at least 50 years of age needed to have an HKA angle of more than 1.3° varus to be included. Patients with ipsilateral total hip arthroplasty, femoral or tibial fracture, osteotomy, or ligamentous repair were excluded. Fifteen alignment parameters were investigated on the sequential full-length X-rays. Moreover, the relationship between the alignment parameters and the Kellgren-Lawrence grade (KL grade) was determined by using linear mixed models. RESULTS: A progressive KL grade is associated with an increase of the HKA (p < 0.001). Mostly, HKA differs due to decrease of the medial tibial plateau (MPTA) angle (0.93°) and an increase of the joint line angle (JLCA) (0.86°). The mLDFA demonstrated the most pronounced changes in the beginning of OA (KL grade 1-2) (p = 0.049). In particular, the MPTA becomes considerably smaller (p = 0.004) in the later stage of OA (KL grade 3). Also, a progressive increase of the JLCA (p < 0.001) is observed upwards of KL grade 3. CONCLUSION: By comparing consecutive full-length X-rays in the same patients, it is possible to define the coronal alignment changes during the arthritic process. The HKA angle increases according the arthritic progression, whereby the most pronounced changes appear at the proximal tibia (MPTA) and at the joint line (JLCA).The alignment changes in varus OA knees can be divided in three stadia: (1) erosion of the distal medial femoral condyle, (2) erosion of the medial tibial plateau, and (3) a progressive increase of the joint line angle. LEVEL OF EVIDENCE: Therapeutic Study, Level III.

The pre-diseased coronal alignment can be predicted from conventional radiographs taken of the varus arthritic knee.

INTRODUCTION: The concept of restoring the constitutional, pre-diseased alignment has gained lots of interest among knee surgeons. Previous attempts to use the contralateral limb to bridge the gap between the arthritic and the constitutional alignment were unsuccessful. We investigated the usability of a mathematical formula to predict the constitutional (pre-diseased) coronal alignment once arthritis has occurred. It is our hypothesis that by using the KL grade, CPAK classification and four radiographic measurements of the arthritic knee, the pre-diseased coronal alignment could be predicted. MATERIAL AND METHODS: Hundred arthritic patients with consecutive X-rays were used to determine a mathematical formula. Five alignment parameters were determined on full-length X-rays: HKA angle, mechanical lateral distal femoral angle (mLDFA), medial proximal tibial angle (MPTA), joint line convergence angle (JLCA) and the tibial joint line angle (TJLA). A refinement of the algorithm was implemented based on a reference cohort, consisted of 250 young adults aged between 20 and 27 years. Moreover, all knees were subdivided based on their CPAK-phenotype and the Kellgren-Lawrence scale (KL scale). An independent arthritic cohort of 289 patients scheduled for primary total knee arthroplasty was included to verify the accuracy of the predicted HKAs (HKAPRED). RESULTS: In CPAK type 1, the HKAPRED was 3.86° varus (STD 1.39) and the HKAYHA was 4.0° varus. In CPAK type 2, the HKAPRED was 1.68° varus (STD 1.95) compared to a HKAYHA of 1.34° (STD 0.81). The average constitutional HKA is not different in both CPAK 1 (p = 0.61) and CPAK 2 (p = 0.25), and the difference in the mean is estimated to be equal to - 0.14 (95CI - 0.68 to 0.40) in CPAK 1 and 0.35 (95CI - 0.06 to 0.75) in CPAK 2. CONCLUSION: Using the KL grade, CPAK classification and four radiographic measurements of the arthritic knee, the pre-diseased coronal alignment can be predicted in 80% of the varus knees with an accuracy of ≤ 0.5°. The predicted HKA (HKAPRED) can be very useful in the current and future clinical practice.

The lateral joint line opening: a radiographic indicative parameter for high grade varus knees.

PURPOSE: It is usually assumed that the severity of varus osteoarthritis (OA) of the knee is correlated with the axis deviation of the limb. Despite this, there is currently no clear radiographic definition to define a so-called 'high degree' varus knee, which is characterized by a pronounced lateral ligamentous laxity. The purpose of this study was to radiographically determine if the lateral joint line opening (LJLO) is an indicative parameter when defining so-called high grade varus knees. METHODS: Two hundred forty Full length radiographs of patients with end-stage varus osteoarthritis who were scheduled for Total knee arthroplasty (TKA) were evaluated. The Hip-knee-ankle-angle (HKA-angle), Joint-line-convergence-angle (JLCA) and the lateral joint line opening were measured. The lateral joint line opening is the shortest distance between the lateral tibial plateau and the deepest point of the lateral femoral condyle. Linear regression models were used to investigate the relationships between the radiographic measurements. RESULTS: Hip-knee-angle-angle, joint-line-conversion-angle, and lateral joint line opening were all positively correlated (p < 0.001). An increase of 1 mm lateral joint line opening causes an increase of 0.6° joint-line-conversion-angle (p = 0.029) below a cut-off point of 4.7 mm. For lateral opening values beyond 4.7 mm, the gradient increased to 1.2 (p < 0.001). A lateral joint line opening of 4.7 mm corresponds to a hip-knee-ankle-angle of 6.0° (95% CI [5.5; 6.5]). CONCLUSION: A lateral joint line opening of more than 5 mm in end-stage OA knees is indicative of increased lateral joint laxity. Those knees can be radiographically classified as so-called 'high-grade' varus knees. LEVEL OF EVIDENCE: Therapeutic study, Level III.

Reactive oxygen species rescue regeneration after silencing the MAPK-ERK signaling pathway in Schmidtea mediterranea.

Despite extensive research on molecular pathways controlling the process of regeneration in model organisms, little is known about the actual initiation signals necessary to induce regeneration. Recently, the activation of ERK signaling has been shown to be required to initiate regeneration in planarians. However, how ERK signaling is activated remains unknown. Reactive Oxygen Species (ROS) are well-known early signals necessary for regeneration in several models, including planarians. Still, the probable interplay between ROS and MAPK/ERK has not yet been described. Here, by interfering with major mediators (ROS, EGFR and MAPK/ERK), we were able to identify wound-induced ROS, and specifically H2O2, as upstream cues in the activation of regeneration. Our data demonstrate new relationships between regeneration-related ROS production and MAPK/ERK activation at the earliest regeneration stages, as well as the involvement of the EGFR-signaling pathway. Our results suggest that (1) ROS and/or H2O2 have the potential to rescue regeneration after MEK-inhibition, either by H2O2-treatment or light therapy, (2) ROS and/or H2O2 are required for the activation of MAPK/ERK signaling pathway, (3) the EGFR pathway can mediate ROS production and the activation of MAPK/ERK during planarian regeneration.

A revisited technique for combined anterior cruciate ligament and anterolateral ligament reconstructions.

A new technique for combined anterior cruciate ligament and anterolateral ligament reconstructions is described. An iliotibial band strip is used as an ALL graft , leaving the distal insertion intact and fix it with a knotless anchor on the femoral origin, after tunneling it under the lateral collateral ligament.

Risk analysis of tunnel collision in combined anterior cruciate ligament and anterolateral ligament reconstructions.

BACKGROUND: To assess the risk of tunnel collision in combined anterior cruciate ligament (ACL) and anterolateral ligament (ALL) reconstructions. METHODS: Three-dimensional (3D) CT reconstructions of 32 knees after transtibial (TT) (N = 16) or anteromedial portal (AMP) (N = 16) ACL reconstruction were used to simulate potential tunnel collision of the femoral ACL tunnel if combined with a virtual ALL reconstruction. The minimal distance between tunnels, the ALL tunnel length, and the lateral femoral condyle (LFC) width were measured. Moreover, the relationship between the ALL tunnel and the intercondylar notch, trochlear groove and posterior femoral cortex was determined. RESULTS: The highest rate of tunnel collision (81%) was observed when the ALL tunnel was aimed at 20° in the coronal plane and 0° in the axial plane. However, by aiming the ALL tunnel at 0° coronal and 40° axial angulation, collision was avoided in all patients and no violation of the trochlea was observed. Tunnel collision rate was significantly higher (P = 0.002) when the ACL tunnel was drilled by the AMP technique. CONCLUSIONS: Risk of tunnel collision was significantly increased when the tunnel was drilled at 0° in the axial plane. Tunnel collision was avoided by aiming the ALL tunnel 40° anteriorly and perpendicular to the anatomical axis of the femur. A more horizontal orientation of the ACL with the AMP technique is a risk factor for tunnel conflicts. CLINICAL RELEVANCE: ALL tunnel orientation needs to be adjusted to avoid tunnel conflicts in combined ACL-ALL reconstructions.

Are Proactive and Reactive Aggression Meaningful Distinctions in Adolescents? A Variable- and Person-Based Approach.

This study was designed to examine whether proactive and reactive aggression are meaningful distinctions at the variable- and person-based level, and to determine their associated behavioral profiles. Data from 587 adolescents (mean age 15.6; 71.6 % male) from clinical samples of four different sites with differing levels of aggression problems were analyzed. A multi-level Latent Class Analysis (LCA) was conducted to identify classes of individuals (person-based) with similar aggression profiles based on factor scores (variable-based) of the Reactive Proactive Questionnaire (RPQ) scored by self-report. Associations were examined between aggression factors and classes, and externalizing and internalizing problem behavior scales by parent report (CBCL) and self-report (YSR). Factor-analyses yielded a three factor solution: 1) proactive aggression, 2) reactive aggression due to internal frustration, and 3) reactive aggression due to external provocation. All three factors showed moderate to high correlations. Four classes were detected that mainly differed quantitatively (no 'proactive-only' class present), yet also qualitatively when age was taken into account, with reactive aggression becoming more severe with age in the highest affected class yet diminishing with age in the other classes. Findings were robust across the four samples. Multiple regression analyses showed that 'reactive aggression due to internal frustration' was the strongest predictor of YSR and CBCL internalizing problems. However, results showed moderate to high overlap between all three factors. Aggressive behavior can be distinguished psychometrically into three factors in a clinical sample, with some differential associations. However, the clinical relevance of these findings is challenged by the person-based analysis showing proactive and reactive aggression are mainly driven by aggression severity.

Renal cells exposed to cadmium in vitro and in vivo: normalizing gene expression data.

Cadmium (Cd) is a toxic metal with a long half-life in biological systems. This half-life is partly as a result of metallothioneins (MTs), metal-binding proteins with a high affinity for Cd. The high retention properties of the kidneys reside in proximal tubular cells that possess transport mechanisms for Cd-MT uptake, ultimately leading to more Cd accumulation. Researchers have studied MT-metal interactions using various techniques including quantitative real-time PCR (qPCR), an efficient tool for quantifying gene expression. Often a poor choice of reference genes, which is represented by their instability and condition dependency, leads to inefficient normalization of gene expression data and misinterpretations. This study demonstrates the importance of an efficient normalization strategy in toxicological research. A selection of stable reference genes was proposed in order to acquire reliable and reproducible gene quantification under metal stress using MT expression as an example. Moreover, in vitro and in vivo setups were compared to identify the influence of toxicological compounds in function of the experimental design. This study shows that glyceraldehyde-3-phosphate dehydrogenase (Gapdh), tyrosine monooxygenase/tryptophan5-monooxygenase activation-protein, zeta polypeptide (Ywhaz) and beta-actin (Actb) are the most stable reference genes in a kidney proximal tubular cell line exposed to moderate and high Cd concentrations, applied as CdCl2 . A slightly different sequence in reference gene stability was found in renal cells isolated from rats in vivo exposed to Cd. It was further shown that three reference genes are required for efficient normalization in this experimental setup. This study demonstrates the importance of an efficient normalization strategy in toxicological research.

The influence of food restriction versus ad libitum feeding of chow and purified diets on variation in body weight, growth and physiology of female Wistar rats.

Ad libitum (AL) supply of standard chow is the feeding method most often used for rodents in animal experiments. However, AL feeding is known to result in a shorter lifespan and decreased health as compared with restricted feeding. Restricted feeding and thus limiting calorie intake prevents many health problems, increases lifespan and can also increase group uniformity. All this leads to a reduced number of animals needed. So-called standard chows are known to be prone to variation in composition. Synthetic foods have a more standard composition, contributing to group uniformity which, like diet reduction, may decrease the number of animals necessary to obtain statistical significance. In this study, we compared the effects of AL versus restricted feeding (25% reduction in food intake) on standard chow versus synthetic food of three different suppliers on body weight (BW), growth, several blood parameters and organ weights in growing female Wistar rats over a period of 61 days. Diet restriction led to a decreased growth and significantly reduced variation in BW and growth as compared with AL feeding. AL feeding on synthetic diets caused a significantly higher BW gain than on chow diets. Due to experimental design, this same effect occurred on food restriction. Blood parameters and organ weights were affected neither by diet type nor by amount. Incidentally, variations were significantly reduced on food restriction versus AL, and on synthetic diets versus chow diets. This study demonstrates that food restriction versus AL feeding leads to a significantly reduced variation in BW and growth, thereby indicating the potential for reduction when applying this feeding schedule.

Isolation and characterization of lectins and lectin-alliinase complexes from bulbs of garlic (Allium sativum) and ramsons (Allium ursinum).

A procedure developed to separate the homodimeric and heterodimeric mannose-binding lectins from bulbs of garlic (Allium sativum L.) and ramsons (Allium ursinum L.) also enabled the isolation of stable lectin-alliinase complexes. Characterization of the individual lectins indicated that, in spite of their different molecular structure, the homomeric and heteromeric lectins resemble each other reasonably well with respect to their agglutination properties and carbohydrate-binding specificity. However, a detailed analysis of the lectin-alliinase complexes from garlic and ramsons bulbs demonstrated that only the heterodimeric lectins are capable of binding to the glycan chains of the alliinase molecules (EC 4.4.1.4). Moreover, it appears that only a subpopulation of the alliinase molecules is involved in the formation of lectin-alliinase complexes and that the complexed alliinase contains more glycan chains than the free enzyme. Finally, some arguments are given that the lectin-alliinase complexes do not occur in vivo but are formed in vitro after homogenization of the tissue.

Developmental Regulation of Lectin and Alliinase Synthesis in Garlic Bulbs and Leaves.

Using a combination of northern blot analysis and sodium dodecyl sulfate-polyacrylamide gel electrophoresis, a detailed study was made of the temporal and spatial regulation of garlic (Allium sativum L.) lectins and alliinase throughout the life cycle of the plant. The two bulb-specific lectins (ASAI and ASAII), which are the most predominant bulb proteins, accumulate exclusively in the developing garlic cloves and progressively disappear when the old clove is consumed by the plant. On the basis of these observations, ASAI and ASAII can be regarded as typical vegetative storage proteins. The leaf-specific lectin (ASAL), on the contrary, is specifically synthesized in young leaves and remains present until withering. Because ASAL is only a minor protein, it probably fulfills a specific function in the plant. Unlike the lectins, alliinase is present in large quantities in bulbs as well as in leaves. Moreover, intact alliinase mRNAs are present in both tissues as long as they contain living cells. The latter observation is in good agreement with the possible involvement of alliinase in the plant's defense against pathogens and/or predators.

Isolation, characterization and molecular cloning of the mannose-binding lectins from leaves and roots of garlic (Allium sativum L.).

Two novel lectins were isolated from roots and leaves of garlic. Characterization of the purified proteins indicated that the leaf lectin ASAL is a dimer of two identical subunits of 12 kDa, which closely resembles the leaf lectins from onion, leek and shallot with respect to its molecular structure and agglutination activity. In contrast, the root lectin ASARI, which is a dimer of subunits of 15 kDa, strongly differs from the leaf lectin with respect to its agglutination activity. cDNA cloning of the leaf and root lectins revealed that the deduced amino acid sequences of ASAL and ASARI are virtually identical. Since both lectins have identical N-terminal sequences the larger Mr of the ASARI subunits implies that the root lectin has an extra sequence at its C-terminus. These results not only demonstrate that virtually identical precursor polypeptides are differently processed at their C-terminus in roots and leaves but also indicate that differential processing yields mature lectins with strongly different biological activities. Further screening of the cDNA library for garlic roots also yielded a cDNA clone encoding a protein composed of two tandemly arrayed lectin domains. Since the presumed two-domain root lectin has not been isolated yet, its possible relationship to the previously described two-domain bulb lectin could not be studied at the protein level.

Lectin and alliinase are the predominant proteins in nectar from leek (Allium porrum L.) flowers.

Analysis of nectar from leek (Allium porrum) flowers by SDS-PAGE revealed the presence of two major polypeptide bands of 50 kDa and 13 kDa, respectively. Using a combination of agglutination tests, enzyme assays and N-terminal sequencing, the polypeptides have been identified as subunits of alliin lyase (alliinase, EC 4.4.1.4) and mannose-binding lectin, respectively. The latter protein is particularly abundant since it represents about 75% of the total nectar protein. Honey produced by bees foraging on flowering leek plants still contains biologically active lectin and alliinase. However, the levels of both proteins are strongly reduced as compared to those in the original nectar. It is evident, therefore, that the lectin as well as the alliinase are inactivated/degraded during the conversion of nectar into honey.

Isolation, characterization and molecular cloning of a leaf-specific lectin from ramsons (Allium ursinum L.).

Lectins were isolated from roots and leaves of ramsons and compared to the previously described bulb lectins. Biochemical analyses indicated that the root lectins AUAIr and AUAIIr are identical to the bulb lectins AUAI and AUAII, whereas the leaf lectin AUAL has no counterpart in the bulbs. cDNA cloning confirmed that the leaf lectin differs from the bulb lectins. Northern blot analysis further indicated that the leaf lectin is tissue-specifically expressed. Sequence comparisons revealed that the ramsons leaf lectin differs considerably from the leaf lectins of garlic, leek, onion and shallot.

The major tuber storage protein of araceae species is a lectin. Characterization and molecular cloning of the lectin from Arum maculatum L.

A new lectin was purified from tubers of Arum maculatum L. by affinity chromatography on immobilized asialofetuin. Although this lectin is also retained on mannose-Sepharose 4B, under the appropriate conditions free mannose is a poor inhibitor of its agglutination activity. Pure preparations of the Arum lectin apparently yielded a single polypeptide band of approximately 12 kD upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis. However, N-terminal sequencing of the purified protein combined with molecular cloning of the lectin have shown that the lectin is composed of two different 12-kD lectin subunits that are synthesized on a single large precursor translated from an mRNA of approximately 1400 nucleotides. Lectins with similar properties were also isolated from the Araceae species Colocasia esculenta (L.) Schott, Xanthosoma sagittifolium (L.) Schott, and Dieffenbachia sequina Schott. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and gel filtration of the different Araceae lectins have shown that they are tetrameric proteins composed of lectin subunits of 12 to 14 kD. Interestingly, these lectins are the most prominent proteins in the tuber tissue. Evidence is presented that a previously described major storage protein of Colocasia tubers corresponds to the lectin.

The bark of Robinia pseudoacacia contains a complex mixture of lectins. Characterization of the proteins and the cDNA clones.

Two lectins were isolated from the inner bark of Robinia pseudoacacia (black locust). The first (and major) lectin (called RPbAI) is composed of five isolectins that originate from the association of 31.5- and 29-kD polypeptides into tetramers. In contrast, the second (minor) lectin (called RPbAII) is a hometetramer composed of 26-kD subunits. The cDNA clones encoding the polypeptides of RPbAI and RPbAII were isolated and their sequences determined. Apparently all three polypeptides are translated from mRNAs of approximately 1.2 kb. Alignment of the deduced amino acid sequences of the different clones indicates that the 31.5- and 29-kD RPbAI polypeptides show approximately 80% sequence identity and are homologous to the previously reported legume seed lectins, whereas the 26-kD RPbAII polypeptide shows only 33% sequence identity to the previously described legume lectins. Modeling the 31.5-kD subunit of RPbAI predicts that its three-dimensional structure is strongly related to the three-dimensional models that have been determined thus far for a few legume lectins. Southern blot analysis of genomic DNA isolated from Robinia has revealed that the Robinia bark lectins are the result of the expression of a small family of lectin genes.

Comparative study of the post-translational processing of the mannose-binding lectins in the bulbs of garlic (Allium sativum L.) and ramsons (Allium ursinum L.).

The biosynthesis and processing of the homodimeric and heterodimeric lectins from the bulbs of garlic (Allium sativum) and ramsons (wild garlic; Allium ursinum) were studied using pulse and pulse-chase labelling experiments on developing bulbs. By combining the results of the in vivo biosynthesis studies and the cDNA cloning of the respective lectins, the sequence of events leading from the primary translation products into the mature lectin polypeptides could be reconstructed. From this it is demonstrated that garlic and ramsons use different schemes of post-translational modifications in order to synthesize apparently similar lectins from totally different precursors. Both the homomeric garlic lectin (ASAII) and its homologue in ramsons (AUAII) are synthesized on the endoplasmic reticulum (ER) as nonglycosylated 13.5 kDa precursors, which, after their transport out of the ER are converted into the mature 12.0 kDa lectin polypeptides by the cleavage of a C-terminal peptide. The heterodimeric garlic lectin ASAI is synthesized on the ER as a single glycosylated precursor of 38 kDa, which after its transport out of the ER undergoes a complex processing which gives rise to two mature lectin subunits of 11.5 and 12.5 kDa. In contrast, both subunits of the heterodimeric ramsons lectin AUAI are synthesized separately on the ER as glycosylated precursors, which after their transport out of the ER are deglycosylated and further processed into the mature lectin polypeptides by the cleavage of a C-terminal peptide.

The monomeric and dimeric mannose-binding proteins from the Orchidaceae species Listera ovata and Epipactis helleborine: sequence homologies and differences in biological activities.

The Orchidaceae species Listera ovata and Epipactis helleborine contain two types of mannose-binding proteins. Using a combination of affinity chromatography on mannose-Sepharose-4B and ion exchange chromatography on a Mono-S column eight different mannose-binding proteins were isolated from the leaves of Listera ovata. Whereas seven of these mannose-binding proteins have agglutination activity and occur as dimers composed of lectin subunits of 11-13 kDa, the eighth mannose-binding protein is a monomer of 14 kDa devoid of agglutination activity. Moreover, the monomeric mannose-binding protein does not react with an antiserum raised against the dimeric lectin and, in contrast to the lectins, is completely inactive when tested for antiretroviral activity against human immunodeficiency virus type 1 and type 2. Mannose-binding proteins with similar properties were also found in the leaves of Epipactis helleborine. However, in contrast to Listera only one lectin was found in Epipactis. Despite the obvious differences in molecular structure and biological activities molecular cloning of different mannose-binding proteins from Listera and Epipactis has shown that these proteins are related and some parts of the sequences show a high degree of sequence homology indicating that they have been conserved through evolution.

Characterization and molecular cloning of mannose-binding lectins from the Orchidaceae species Listera ovata, Epipactis helleborine and Cymbidium hybrid.

Mannose-binding lectins were purified from the leaves of three Orchidaceae species, namely Listera ovata (twayblade), Epipactis helleborine (broad-leaved helleborine) and Cymbidium hybrid, using affinity chromatography on Mannose - Sepharose-4B. Apparently, the Orchidaceae lectins are dimeric proteins composed of lectin subunits of 12-13 kDa. All of the isolated lectins exhibit exclusive specificity towards mannose. A cDNA library constructed from poly(A) rich RNA isolated from leaves of L. ovata was screened for cDNA clones encoding the lectin using colony hybridization. Since N-terminal sequence analysis of the twayblade lectin revealed some sequence similarity to the previously cloned mannose-binding lectin Hippeastrum hybrid (amaryllis) ovaries, the amaryllis lectin cDNA clone was used as a probe to screen the L. ovata library. Subsequently, the cDNA clone encoding the L. ovata lectin was used to screen the cDNA libraries from the taxonomically related orchid species Cymbidium hybrid and E. helleborine. Sequence analysis of the lectin cDNA clones from different Orchidaceae species revealed approximately 50% sequence similarity both at the nucleotide and amino acid level. The Orchidaceae lectins are apparently translated from mRNAs consisting of approximately 800 nucleotides. The primary translation products are preproproteins which are converted into the mature lectins following post-translational modifications. Southern blot analysis of genomic DNA has shown that the lectins are most probably encoded by a family of closely related genes which is in good agreement with the sequence heterogeneity found between different lectin cDNA clones of one species.