Drought effects on the dynamics of leaf production and senescence in field-grown Medicago arborea and Medicago citrina

Forage shrub production in the Mediterranean region is frequently limited by soil water availability. To study plant responses to water deficit under such conditions is important for improving crop management and for selecting better yielding forage shrub species. Pre-dawn leaf water potential (Ψ_pd), plant leaf area (PLA), leaf area per stem (LA_s), leaf appearance rate (LAR₁;), leaf senescence rate (LSR), individual leaf area (LA) and maximal leaf elongation rate (LER) were studied throughout the year for Medicago arborea (MA) and Medicago citrina (MC) under irrigated (control) and low rainfall field conditions, at the experimental field site of the University of the Balearic Islands in Spain. With irrigation, the highest LA and LER were observed in autumn and spring and the lowest in winter and summer. LAR; was similar for both species in autumn and winter. Throughout the spring, LAR₁ was higher for MC compared to MA. PLA was similar for both species during the autumn, winter and spring seasons; however, during the summer PLA of MA was significantly reduced by 53%. This decline was attributed to higher leaf senescence during seed maturity. As a consequence, MC maintained higher leaf area (∼ 5 m² plant⁻¹) than MA (3 m² plant⁻¹). Under natural field conditions, soil water deficit increased from February to late August. The main effect of water stress was a marked reduction in LAR₁, LA and LER reflected in lower LA_s and PLA. Leaf area was severely reduced for both species during the summer, but much more intensively in MA, which developed full leaf senescence. Thus, MC maintained higher PLA than MA (0.5 m² compared to 0.0 m²). Throughout the year, but especially in the driest months, MC was superior to MA in leaf growth parameters and PLA.     

Estimating the needle area from geometric measurements: application of different calculation methods to Norway spruce

Different calculation methods, based on needle geometry, for estimating both projected area (PLA) and total surface area (TLA) of foliage in Norway spruce [Picea abies (L.) Karst.] were compared. Seventy-eight shoots of four age classes were sampled from both the basal and top thirds of crowns. Three dimensions (the length, minor and major diameters) of needles were taken, and the needle shape was approximated to a parallelepiped or ellipsoid. There was a perfect coincidence of the measured and estimated values of PLA calculated as the width of the needle projection multiplied by needle length, and corrected for needle taper (method III), or when the needle projection was treated as a rectangle joined with half-ellipses at both ends (method IV). The most reliable estimations of TLA resulted from treating the needle sides as faces of the parallelepiped tapering at their ends in the form of half-ellipses. The ratio of TLA to PLA ranged from 2.2 to 4.0 depending on the needle morphology. Needle minor diameter (anatomical width; D ₁) was found to be a better morphological index of the spruce foliage than needle flatness, i.e. the ratio of major to minor diameter. Expressing the factor for converting PLA to TLA as a function of D ₁ considerably improved the precision of the estimates. Close relationships were established between specific leaf area, expressed on both a projected area (SLA_P) and total surface area basis (SLA_T), needle dry weight (R ² was 0.799 and 0.852, respectively) and minor diameter of needles (R ² was 0.701 and 0.554, respectively). Received: 14 April 1998 / Accepted: 23 September 1999     

Mechanistic understanding of Phenyllactic acid mediated inhibition of quorum sensing and biofilm development in Pseudomonas aeruginosa

Pseudomonas aeruginosa depends on its quorum sensing (QS) system for its virulence factors’ production and biofilm formation. Biofilms of P. aeruginosa on the surface of indwelling catheters are often resistant to antibiotic therapy. Alternative approaches that employ QS inhibitors alone or in combination with antibiotics are being developed to tackle P. aeruginosa infections. Here, we have studied the mechanism of action of 3-Phenyllactic acid (PLA), a QS inhibitory compound produced by Lactobacillus species, against P. aeruginosa PAO1. Our study revealed that PLA inhibited the expression of virulence factors such as pyocyanin, protease, and rhamnolipids that are involved in the biofilm formation of P. aeruginosa PAO1. Swarming motility, another important criterion for biofilm formation of P. aeruginosa PAO1, was also inhibited by PLA. Gene expression, mass spectrometric, functional complementation assays, and in silico data indicated that the quorum quenching and biofilm inhibitory activities of PLA are attributed to its ability to interact with P. aeruginosa QS receptors. PLA antagonistically binds to QS receptors RhlR and PqsR with a higher affinity than its cognate ligands N-butyryl-l-homoserine lactone (C₄–HSL) and 2-heptyl-3,4-dihydroxyquinoline (PQS; Pseudomonas quinolone signal). Using an in vivo intraperitoneal catheter-associated medaka fish infection model, we proved that PLA inhibited the initial attachment of P. aeruginosa PAO1 on implanted catheter tubes. Our in vitro and in vivo results revealed the potential of PLA as anti-biofilm compound against P. aeruginosa.

     

Biological characterization of D-lactate dehydrogenase responsible for high-yield production of D-phenyllactic acid in Sporolactobacillus inulinus

PLA (3-D-phenyllactic acid) is an ideal antimicrobial and immune regulatory compound present in honey and fermented foods. Sporolactobacillus inulinus is regarded as a potent D-PLA producer that reduces phenylpyruvate (PPA) with D-lactate dehydrogenases. In this study, PLA was produced by whole-cell bioconversion of S. inulinus ATCC 15538. Three genes encoding D-lactate dehydrogenase (d-ldh1, d-ldh2, and d-ldh3) were cloned and expressed in Escherichia coli BL21 (DE3), and their biochemical and structural properties were characterized. Consequently, a high concentration of pure D-PLA (47 mM) was produced with a high conversion yield of 88%. Among the three enzymes, D-LDH1 was responsible for the efficient conversion of PPA to PLA with kinetic parameters of Km (0.36 mM), k_cat (481.10 s⁻¹), and k_cat/Km (1336.39 mM⁻¹ s⁻¹). In silico structural analysis and site-directed mutagenesis revealed that the Ile307 in D-LDH1 is a key residue for excellent PPA reduction with low steric hindrance at the substrate entrance. This study highlights that S. inulinus ATCC 15538 is an excellent PLA producer, equipped with a highly specific and efficient D-LDH1 enzyme.     

Poly-l-lysines and poly-l-arginines induce leakage of negatively charged phospholipid vesicles and translocate through the lipid bilayer upon electrostatic binding to the membrane

Poly-l-lysines (PLL) and poly-l-arginines (PLA) of different polymer chain lengths interact strongly with negatively charged phospholipid vesicles mainly due to their different electrical charges. 1-Palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol (POPG), 1,2-dipalmitoyl-sn-glycero-3-phosphoglycerol (DPPG) and their mixtures (1/1 mol/mol) with the respective phosphatidylcholines of equivalent chain length were chosen as model membrane systems that form at room temperature either the fluid L_α or the gel phase L_β lipid bilayer membranes, respectively. Leakage experiments revealed that the fluid POPG membranes are more perturbed compared to the gel phase DPPG membranes upon peptide binding. Furthermore, it was found that pure PG membranes are more prone to release the vesicle contents as a result of pore formation than the lipid mixtures POPG/POPC and DPPG/DPPC. For the longer polymers (≥ 44 amino acids) maximal dye-release was observed when the molar ratio of the concentrations of amino acid residues to charged lipid molecules reached a value of R_P = 0.5, i.e. when the outer membrane layer was theoretically entirely covered by the polymer. At ratios lower or higher than 0.5 leakage dropped significantly. Furthermore, PLL and PLA insertions and/or translocations through lipid membranes were analyzed by using FITC-labeled polymers by monitoring their fluorescence intensity upon membrane binding. Short PLL molecules and PLA molecules of all lengths seemed to translocate through both fluid and gel phase lipid bilayers. Comparison of the PLL and PLA fluorescence assay results showed that PLA interacts stronger with phospholipid membranes compared to PLL. Isothermal titration calorimetry (ITC) measurements were performed to give further insight into these mechanisms and to support the findings obtained by fluorescence assays. Cryo-transmission electron microscopy (cryo-TEM) was used to visualize changes in the vesicles' morphology after addition of the polypeptides.     

Enrichment of pinolenic acid from pine nut oil via lipase-catalyzed ethanolysis with an immobilized Candida antarctica lipase

Abstract

Pinolenic acid (PLA) enrichment as an ethyl ester from pine nut oil was successfully accomplished in a batch reactor by lipase-catalyzed ethanolysis using Novozym 435 lipase from Candida antarctica as a biocatalyst. PLA is predominantly an sn-3 substituent of the pine nut oil triacylglycerol (TAG), where it accounts for about 39 mol% of the fatty acids esterified at that position. In the presence of ethanol, Novozym 435 exhibited sn-3 regiospecificity with respect to the TAG of pine nut oil. The effect of the molar ratio of reactants on PLA enrichment by ethanolysis was investigated. The molar ratios of pine nut oil to ethanol were varied from 1:20 to 1:100. A fatty acid ethyl ester (FAEE) fraction with higher PLA content was obtained in the early stage of the reaction, although the yield of PLA was small. However, the PLA content of the FAEEs decreased with increasing reaction time, while the yield of PLA increased. The molar ratio of pine nut oil to ethanol that produced the optimum content and yield of PLA in FAEEs was 1:80.     

Mechanisms of neutrophil-induced DNA damage in respiratory tract epithelial cells

Reactive oxygen species (ROS) released by neutrophils have been suggested to play an important role in cancer development. Since the mechanisms underlying this effect in the respiratory tract are still unclear, we evaluated DNA damage induced by neutrophils in respiratory tract epithelial cells in vitro and in vivo. For in vitro studies, rat lung epithelial cells (RLE) were co-incubated with activated neutrophils, neutrophil-conditioned medium, or hydrogen peroxide. For in vivo studies, we considered the human nose as a target organ, comparing neutrophilic inflammation in the nasal lavage fluid with the oxidative DNA lesion 8-hydroxydeoxyguanosine (8-OHdG) in epithelial cells obtained by nasal brush. Our in vitro data show that human neutrophils are able to induce both 8-OHdG and strand breaks in DNA from RLE cells. Our data also suggest that DNA damage induced by neutrophils is inhibited when neutrophil-derived H₂O₂ is consumed by myeloperoxidase. In contrast, in the nose no association between neutrophil numbers and 8-OHdG was found. Therefore, it remains unclear whether neutrophils pose a direct genotoxic risk for the respiratory tract epithelium during inflammation, and more in vivo studies are needed to elucidate the possible association between neutrophils and genotoxicity in the lung.     

Histological development of the long‐snouted seahorse Hippocampus guttulatus during ontogeny

The objective of the present study was to describe histological development of the European long‐snouted seahorse Hippocampus guttulatus, to increase understanding of the biology and physiology of the species. Most vital organs were present in juveniles by the time of their release from the male's pouch. Digestive tract specialization occurred at 89 effective day‐degrees (D°_eff), corresponding to 15 days post partum (dpp), with development of the first intestinal loop and mucosal folding. At 118 D°_eff (20 dpp), lipids were being mobilized from the liver and oocytes attained the perinuclear stage. The fovea emerged at 177 D°_eff (30 dpp), contemporaneous with the shift from pelagic to benthic behaviour in juveniles. At this stage, the most interesting feature was the formation of the second intestinal loop. Male gonads were never observed during the study (from 0 to 354 D°_eff; 0–60 dpp), but the first oogonia were present at 30 D°_eff (5 dpp). In 354 D°_eff (60 dpp) juveniles, oocytes were observed in a cortical alveoli stage, indicating maturity. Low digestive efficiency was observed at early stages, which was due to a poorly developed gastrointestinal tract and an immature digestive tract prior to 89 D°_eff. The present study demonstrates that approximately 89 and 177 D°_eff represent two important transitional stages in the early development of H. guttulatus. At a temperature of approximately 19 ± 1°C and an age of 1 month (177 D°_eff), main organs were fully functional, suggesting that the adult phenotype was largely established by that age, with females becoming mature at the age of 2 months (354 D°_eff).     

Toxicity of phospholipases A<Subscript>2</Subscript> D49 (6-1 and 6-2) and K49 (Bj-VII) from <Emphasis Type="Italic">Bothrops jararacussu</Emphasis> venom

Purified phospholipase A₂ (PLA2) enzymes from Bothrops jararacussu snake venom were examined to evaluate NIH 3T3 and COS7 fibroblast cytotoxicity, as well as muscle myotoxic and inflammatory activities. Separation of fractions Bj-VII (from BthTX-I; a Lys49 PLA2 homolog) and 6-1 and 6-2 (from BthTX-II; an Asp49 PLA2) from B. jararacussu snake venom by SDS-PAGE in tricine buffer in the absence and presence of dithiothreitol revealed a homodimer with an estimated molecular mass of ∼30 kDa (monomer mass ∼15 kDa). This finding indicates that these toxins form dimeric complexes—a previously reported tendency among PLA2s. These toxins were assayed for viability with the MTT assay, which is used to examine the effects of phospholipases on the mitochondrial viability of cells. The toxins were also assayed for cytolysis of the fibroblast cell lines NIH 3T3 and COS7 by quantification of lactate dehydrogenase released into the medium. The results indicate that the PLA2s 6-1, 6-2 and the Bj-VII PLA2 homolog studied here induce moderate footpad edema and local myotoxicity. Moreover, exposure to these phospholipases led to a reduction in fibroblast viability; at the 1 μM dose of PLA2 tested, a reduction of 50% in cell viability was observed. The present findings indicate that the inflammatory activity observed in envenomation may be correlated with the cytotoxicity observed in fibroblasts.     

Interisland Evolution of <Emphasis Type="Italic">Trimeresurus flavoviridis</Emphasis> Venom Phospholipase A<Subscript>2</Subscript> Isozymes

Abstract Trimeresurus flavoviridis snakes inhabit the southwestern islands of Japan. A phospholipase A₂ (PLA₂), named PL-Y, was isolated from Okinawa T. flavoviridis venom and its amino acid sequence was determined from both protein and cDNA. PL-Y was unable to induce edema. In contrast, PLA-B, a PLA₂ from Tokunoshima T. flavoviridis venom, which is different at only three positions from PL-Y, is known to induce edema. A new PLA₂, named PLA-B′, which is similar to PLA-B, was cloned from Amami-Oshima T. flavoviridis venom gland. Three T. flavoviridis venom basic [Asp⁴⁹]PLA₂ isozymes, PL-Y (Okinawa), PLA-B (Tokunoshima), and PLA-B′ (Amami-Oshima), are identical in the N-terminal half but have one to four amino acid substitutions in the β1-sheet and its vicinity. Such interisland sequence diversities among them are due to isolation in the different environments over 1 to 2 million years and appear to have been brought about by natural selection for point mutation in their genes. Otherwise, a major PLA₂, named PLA2, ubiquitously exists in the venoms of T. flavoviridis snakes from the three islands with one to three synonymous substitutions in their cDNAs. It is assumed that the PLA2 gene is a prototype among T. flavoviridis venom PLA₂ isozyme genes and has hardly undergone nonsynonymous mutation as a principal toxic component. Phylogenetic analysis based on the amino acid sequences revealed that T. flavoviridis PLA₂ isozymes are clearly separated into three groups, PLA2 type, basic [Asp⁴⁹]PLA₂ type, and [Lys⁴⁹]PLA₂ type. Basic [Asp⁴⁹]PLA₂-type isozymes may manifest their own particular toxic functions different from those of the isozymes of the PLA2 type and [Lys⁴⁹]PLA₂ type.     

The response of plants to elevated CO2

Summary Four coexisting annual plant species were grown in competition at three levels of CO₂ (300, 600, and 1,200 ppm) and two levels of soil moisture (moist and dry). Plant height was higher at high CO₂ concentrations for the three C₃ species but not for the C₄ species (Amaranthus retroflexus). Total community biomass increased with increasing CO₂ at both soil moisture levels. The contribution of each species to total community biomass was influenced by CO₂ concentration. The effects were especially pronounced for Polygonum pensylvanicum which contributed more to community production as CO₂ and soil moisture increased. Amaranthus behaved in exactly the reverse way; it did best under ambient CO₂ and dry soil moisture conditions. The results suggest that changes in competitive interactions and community structure will occur with the anticipated rise in global CO₂ concentration.     

MOULT IN FIVE SPECIES OF CORVIDAE IN BRITAIN

This paper presents objective methods for measuring moult and uses them to describe the pattern and rate of the complete moult as a component of the annual cycle in Corvus corone, C. frugilegus, C. monedula, Pica pica and Garrulus glandarius in Britain. The basis of the methods used to measure the rate of moult was the dry weight of the whole plumage and its component tracts and feathers. Within whole tracts of flight feathers showing diversity of size, the longer feathers grew in length a little faster than the others by accumulating feather material very much faster. Thus, for the flight feathers at least length by itself was a poor indicator of size, but, if first related to weight, could be used to predict weight. Hence, to enable realistic comparisons to be made between different flight feathers and tracts of flight feathers on a given individual bird or on individuals of different species, a ‘units’ system was created which would take account of such variations in size. This information provided a standard against which the growth of new flight feathers in a specimen collected in the field could be measured. The progress of moult in each tract was indicated by the sum of the units. In addition, for both the flight feathers and all other tracts, the duration of moult was measured from the start and finish in each tract as indicated by the proportion of birds having either growing or entirely fully-grown feathers in each tract. In the annual cycle body weight and abdominal fat weight were at a maximum in mid-winter and at a minimum in mid-summer in Corvus; variations in these items were apparently absent in G. glandarius. In Corvus probably only post-second-year birds breed, but in P. pica some second-year individuals do so as well; also, increase in testes size in second-year males was greatest in P. pica. The patterns of the relative seasonal timing of the start and finish of moult in each tract were most similar in the three Corvus species, in which moult in the whole plumage began in the primary tract and finished in tracts on the body; P. pica differed principally in the extended growth of the secondary tract; G. glandarius differed in that moult began in the dorsal tract. By summing the data from individual tracts, a measure of the rate of growth in the whole plumage was obtained: all five species moulted at essentially the same time of year. In the primary, secondary, tertiary and rectricial tracts the seasonal accumulation of dry weight of feather material in each tract followed a sigmoid pattern; in the alula tract dry weight increased most rapidly at the beginning. These patterns were correlated with the numbers of feathers growing at different stages in each tract. The primary tract accumulated dry matter at the highest relative rate, while the rectricial, secondary, tertiary and alula tracts did so at successively lower rates. The maximum relative rate of increase in dry weight in any given tract of flight feathers was fairly similar in all five species, but was possibly related inversely to body weight. Maximum absolute rates of increase in dry tract weight were also calculated. During the springtime period when non-moulting and moulting birds were present simultaneously in the population, no difference could be detected between these groups in any species in body and fat weights; in individuals obtained together with evidence of reproductive activity, few had begun primary moult, but in post-second-year C. frugilegus with branchers, males began before females; in C. corone and C. frugilegus testes weight was lower in moulting than in non-moulting birds. In conclusion, some aspects of moult for further research are suggested.     

Protective effect of N-(p-amylcinnamoyl) anthranilic acid,phospholipase A2 enzyme inhibitor,and transient receptor potential melastatin-2 channel blocker against renal ischemia–reperfusion injury

The production of reactive oxygen species and inflammatory events are the underlying mechanisms of ischemia-reperfusion injury (IRI). It was determined that transient receptor potential melastatin-2 (TRPM2) channels and phospholipase A₂ (PLA ₂) enzymes were associated with inflammation and cell death. In this study, we investigated the effect of N-( p-amylcinnamoyl) anthranilic acid (ACA), a TRPM2 channel blocker, and PLA ₂ enzyme inhibitor on renal IRI. A total of 36 male Sprague-Dawley rats were divided into four groups: control, ischemia-reperfusion (I/R), I/R + ACA 5 mg, I/R + ACA 25 mg. In I/R applied groups, the ischemia for 45 minutes and reperfusion for 24 hours were applied bilaterally to the kidneys. In the I/R group, serum levels of the blood urea nitrogen (BUN), creatinine, cystatin C (CysC), kidney injury molecule-1 (KIM-1), neutrophil gelatinase-associated lipocalin (NGAL), and interleukin-18 increased. On histopathological examination of renal tissue in the I/R group, the formation of glomerular and tubular damage was seen, and it was detected that there was an increase in the levels of malondialdehyde (MDA), caspase-3, total oxidant status (TOS), and oxidative stress index (OSI); and there was a decrease in total antioxidant capacity (TAC) and catalase enzyme activity. ACA administration reduced serum levels of BUN, creatinine, CysC, KIM-1, NGAL, interleukin-18. In the renal tissue, ACA administration reduced histopathological damage, levels of caspase-3, MDA, TOS, and OSI; and it increased the level of TAC and catalase enzyme activity. It has been shown with the histological and biochemical results in this study that ACA is protective against renal IRI.     

微塑料对稻田土壤温室气体排放和微生物群落的影响

微塑料因在土壤环境中广泛存在及其潜在的生态风险而受到越来越多的关注。微塑料的赋存会改变土壤理化性质,并对土壤微生物群落及其驱动的生物地球化学过程产生影响,而相关研究尚处于起步阶段。可生物降解塑料作为传统塑料的替代品,越来越多地应用于农业活动,并释放到土壤中。然而,可生物降解微塑料对土壤微生物特性产生影响的研究鲜有报道。基于此,本试验以我国三江平原水稻田土壤为研究对象,选取了2种常见的微塑料为试验材料,分别为传统型微塑料聚丙烯(Polypropylene,PP)和可降解微塑料聚乳酸(Polylactic acid,PLA),进行了为期41d的微宇宙培养实验,旨在分析不同浓度与类型的微塑料对土壤可溶性有机碳(Dissolved Organic Carbon,DOC)含量及官能团特征、温室气体排放以及微生物群落结构的差异性影响。结果表明,传统型微塑料PP与可降解微塑料PLA添加均对土壤理化性质与微生物群落产生显著影响。其中,微塑料添加大体上增加了土壤DOC含量,PLA的促进作用较为明显,且增加含量与微塑料添加量呈正相关;PP和PLA均影响土壤DOC分子结构,削弱了土壤团聚化程度并促进了大分子量DOC化合物的生成;微塑料的添加促进土壤CH₄排放,而有效抑制了土壤CO₂排放;微塑料显著改变了土壤细菌和真菌群落的丰富度与多样性。相关分析结果表明,土壤CO₂累计排放量与芳香族化合物结构及疏水性等官能团特征、变形菌门(Proteobacteria)与放线菌门(Actinobacteria)均呈显著正相关关系。以上结果表明,微塑料添加改变了土壤DOC含量及官能团特征与微生物环境,进而影响土壤温室气体排放。本研究为今后微塑料对土壤地球化学和微生物特性的影响研究提供了科学的思路,同时也有助于评估微塑料对土壤生态系统的生态风险。     

Retina-specific LDH isozyme in blueback herring, Alosa aestivalis (Mitchell), and alewife, Aiosa pseudoharengus (Wilson)

The retina of 390 Alosa aestivalis and 410 Alosa pseudoharengus have been examined by means of starch-gel electrophoresis. The retina-specific E₄ isozyme has been found to occur in all the fish examined. This study demonstrates for the first time that the E₄ isozyme occurs in A. aestivalis. Because the E₄ isozyme is not polymorphic and has an identical mobility in A. pseudoharengus and in A. aestivalis it is neither suitable for use as a species identification characteristic nor a population marker. Alosa aestivalis and Alosa pseudoharengus are two commercially important ana-dromous species of fish in New Brunswick, Canada. These two species may occur together in the same spawning runs but w^rhile A. pseudoharengus has a wide distribution along the East coast of North America (Leim & Scott, 1966) A. aestivalis occurs in a very limited area in Canada where it is at the northern limit of its range and because of increasing threat of pollution has been listed by McAllister (1970) as one of 17 endangered species. These two species of fish are morphologically very similar and can only be separated by the colour of the abdominal peritoneum (Leim & Scott, 1966). McKenzie (1973) has compared these two species by means of protein electrophoresis and found that while the muscle myogen patterns were species specific, the LDH patterns were the same in both species. He described these two species as five isozyme fish showing the LDH isozymes A₄, A₃B, A₂B₂, AB₃ and B₄. Since Horowitz & Whitt (1972) have reported the presence of the E₄ isozyme in the retina of some teleosts including. A. pseudoharengus but not including A. aestivalis, I considered it worth-while to re-examine A. aestivalis and A. pseudoharengus to find out whether A. aestivalis possessed this isozyme and, if so, whether the mobility of the isozyme could be used as a species identification characteristic and as a population marker. The fish used in this study were collected from five different locations during the 1971 spring migration period and held deep frozen for eight months before they were examined. They were identical to the specimens used for the study reported by McKenzie (1973) where the collection dates, numbers of fish and geographic locations are given. One eyeball from each of 390 A. aestivalis and 410 A. pseudoharengus was removed. Each eyeball was homogenized in 10 drops of distilled water and allowed to stand for one hour at 4^oC. The samples were then centrifuged for 10 min at 12 000 g. The supernatants were used immediately for vertical starch-gel electrophoresis. The apparatus used was that described by Boyer & Hiner (1963). The conditions of electrophoresis were the same as used by Saunders & McKenzie (1971). The LDH bands were stained by the deposition of blue formazan dyes in the regions of LDH activity. The stain formula and details of methods are given in Whitt (1970). The LDH isozyme patterns of all the fish examined were identical. The location of the isozymes A₄, A₃B, A₂B₂, AB₃ and B₄ are indicated in Fig. 1. A₄ is abundant in muscle while B₄ is abundant in heart. Because the LDH subunits assemble preferentially into homodimeric pairs before forming tetramers (Markert & Ursprung, 1971). A₄. A₂B₂ and B₄ show up in electropherograms as strong bands while A₃B and AB₃ show up as weak bands. The retina-specific isozyme E₄ is shown between B₄ and AB₃. Whitt (1970) has already demonstrated that A. pseudoharengus is a five isozyme fish. This has been confirmed by McKenzie (1973) who also compared both A. pseudoharengus and A. aestivalis and found these five isozymes had identical mobilities in both species of fish. The present study demonstrates for the first time that the retina-specific E₄ occurs in A. aestivalis where it has the same electro-phoretic mobility as that of A. pseudoharengus. The patterns are the same and do not appear to vary with geographic origin of the fish. The reason why the presence of the E₄ isozyme was demonstrated in the present study and not in the previous one (McKenzie, 1973) is because of the method used. In that study the migration length was not long enough to sufficiently separate the enzymes. In the present study vertical starch-gel electrophoresis which allows for long migration distances was used. As has already been shown for the A-B isozymes (McKenzie, 1973), the E₄ isozyme is not polymorphic in these two species of fish. It therefore has no use as apopulation marker. Because the E₄ isozyme has an identical mobility in both species of fish, it cannot be used as a species identification characteristic.     

Electron microscopy of α2-macroglobulin with a thiol ester bound ligand

In order to covalently bind the hydrolyzed thiol ester groups of the human α₂-macroglobulin (α2M) transformed by methylamine, the phospholipase A2 (PLA2), a small enzyme (M_r = 13 000) from Naja nigricollis snake venom was activated by succinimidyl 4-(maleimidomethyl)cyclohexane-1-carboxylate (SMCC). Average images determined from electron micrographs of the methylamine-transformed α2M, with and without activated PLA2, were determined by image processing and compared. A localization of the PLA2 was achieved by subtracting the average image of α2M transformed by methylamine from that containing PLA2. The results are consistent with previous work showing the central localization of chymotrypsin trapped in α2M. They also suggest that the four thiol esters are located near the center of the α2M. molecule.     

Effect of soil drying on growth,biomass allocation and leaf gas exchange of two annual grass species

Influence of short-term water stress on plant growth and leaf gas exchange was studied simultaneously in a growth chamber experiment using two annual grass species differing in photosynthetic pathway type, plant architecture and phenology:Triticum aestivum L. cv. Katya-A-1 (C₃, a drought resistant wheat cultivar of erect growth) andTragus racemosus (L.) All. (C₄, a prostrate weed of warm semiarid areas). At the leaf level, gas exchange rates declined with decreasing soil water potential for both species in such a way that instantaneous photosynthetic water use efficiency (PWUE, mmol CO₂ assimilated per mol H₂O transpired) increased. At adequate water supply, the C₄ grass showed much lower stomatal conductance and higher PWUE than the C₃ species, but this difference disappeared at severe water stress when leaf gas exchange rates were similarly reduced for both species. However, by using soil water more sparingly, the C₄ species was able to assimilate under non-stressful conditions for a longer time than the C₃ wheat did. At the whole-plant level, decreasing water availability substantially reduced the relative growth rate (RGR) ofT. aestivum, while biomass partitioning changed in favour of root growth, so that the plant could exploit the limiting water resource more efficiently. The change in partitioning preceded the overall reduction of RGR and it was associated with increased biomass allocation to roots and less to leaves, as well as with a decrease in specific leaf area. Water saving byT. racemosus sufficiently postponed water stress effects on plant growth occurring only as a moderate reduction in leaf area enlargement. For unstressed vegetative plants, relative growth rate of the C₄ T. racemosus was only slightly higher than that of the C₃ T. aestivum, though it was achieved at a much lower water cost. The lack of difference in RGR was probably due to growth conditions being relatively suboptimal for the C₄ plant and also to a relatively large investment in stem tissues by the C₄ T. racemosus. Only 10% of the plant biomass was allocated to roots in the C₄ species while this was more than 30% for the C₃ wheat cultivar. These results emphasize the importance of water saving and high WUE of C₄ plants in maintaining growth under moderate water stress in comparison with C₃ species.     

Looking for Organization Patterns of Highly Expressed Genes: Purine-Pyrimidine Composition of Precursor mRNAs

We analyzed precursor messenger RNAs (pre-mRNAs) of 12 eukaryotic species. In each species, three groups of highly expressed genes, ribosomal proteins, heat shock proteins, and amino-acyl tRNA synthetases, were compared with a control group (randomly selected genes). The purine-pyrimidine (R-Y) composition of pre-mRNAs of the three targeted gene groups proved to differ significantly from the control. The exons of the three groups tested have higher purine contents and R-tract abundance and lower abundance of Y-tracts compared to the control (R-tract—tract of sequential purines with R _n ≥ 5; Y-tract—tract of sequential pyrimidines with Y _n ≥ 5). In species widely employing “intron definition” in the splicing process, the Y content of introns of the three targeted groups appeared to be higher compared to the control group. Furthermore, in all examined species, the introns of the targeted genes have a lower abundance of R-tracts compared to the control. We hypothesized that the R-Y composition of the targeted gene groups contributes to high rate and efficiency of both splicing and translation, in addition to the mRNA coding role. This is presumably achieved by (1) reducing the possibility of the formation of secondary structures in the mRNA, (2) using the R-tracts and R-biased sequences as exonic splicing enhancers, (3) lowering the amount of targets for pyrimidine tract binding protein in the exons, and (4) reducing the amount of target sequences for binding of serine/arginine-rich (SR) proteins in the introns, thereby allowing SR proteins to bind to proper (exonic) targets. (Reviewing Editor: Dr. Axel Meyer)     

Phospholipase A2 in cnidaria

Phospholipase A2 (PLA2) is an enzyme present in snake and other venoms and body fluids. We measured PLA2 catalytic activity in tissue homogenates of 22 species representing the classes Anthozoa, Hydrozoa, Scyphozoa and Cubozoa of the phylum Cnidaria. High PLA2 levels were found in the hydrozoan fire coral Millepora sp. (median 735 U/g protein) and the stony coral Pocillopora damicornis (693 U/g) that cause skin irritation upon contact. High levels of PLA2 activity were also found in the acontia of the sea anemone Adamsia carciniopados (293 U/g). Acontia are long threads containing nematocysts and are used in defense and aggression by the animal. Tentacles of scyphozoan and cubozoan species had high PLA2 activity levels: those of the multitentacled box jellyfish Chironex fleckeri contained 184 U/g PLA2 activity. The functions of cnidarian PLA2 may include roles in the capture and digestion of prey and defense of the animal. The current observations support the idea that cnidarian PLA2 may participate in the sting site irritation and systemic envenomation syndrome resulting from contact with cnidarians.