Comparison of neutral lipid profile of various trilaminar outer cell wall (TLS)-containing microalgae with emphasis on algaenan occurrence

The neutral lipid profiles of nine species of thin trilaminar outer wall (TLS)-containing freshwater and marine microalgae from the class of Chlorophyceae were studied with emphasis on the relationship between the lipid content and the occurrence of insoluble non-hydrolysable biopolymer (i.e. algaenan). All the freshwater microalgae produce a highly aliphatic algaenan. In sharp contrast, no algaenan was isolated from the two marine microalgae, Chlorella marina and Chlorella minutissima marina, supporting the absence of a close relationship between the presence of TLS and the occurrence of algaenan. High molecular weight straight-chain hydrocarbons (C23-C29) were identified in most of the algaenan-producing microalgae and in the algaenan-devoid C. minutissima marina, whereas only low molecular weight hydrocarbons were detected in algaenan-producing Scenedesmus subspicatus and in algaenan-devoid C. marina. Sterols, phytol and fatty alcohols were the major constituents of the polar fraction of the neutral lipids of all the microalgae investigated. High molecular weight saturated or mono-unsaturated alcohols were detected in C. emersonii and in all the microalgae belonging to the genus Scenedesmus. High amounts of saturated C30 and C32 alpha,omega-diols were also detected in S. subspicatus, S. armatus and S. pannonicus. Three classes of lipids were encountered in very small amounts in the medium polarity fraction of the neutral lipids of the microalgae investigated: (i) Monoesters composed predominantly of saturated C16 or C18 fatty acids and saturated C8, C16 or C18 alcohols and (ii) long-chain methyl ketones from C25 to C31 were detected in several species and (iii) methyl esters of fatty acids ranging from C16 to C28 were identified in all the microalgae. Attempts to use the neutral lipid composition and particularly the unusual long-chain lipids, as specific indicators of the occurrence of algaenan in TLS-containing microalgae were unsuccessful.     

Unveiling antimicrobial activity of microalgae Chlorella sorokiniana (UKM2), Chlorella sp. (UKM8) and Scenedesmus sp. (UKM9)

Microalgae represent promising sources of bioactive compounds for pharmaceutical and industrial applications. The emergence of antibiotic resistant bacteria leads to the need to explore new cost-effective, safe, and potent bioactive compounds from the microalgae. This study aimed to investigate the potential of local microalgae for their antimicrobial properties and bioactive compounds. Three local microalgae namely Chlorella sorokiniana (UKM2), Chlorella sp. UKM8, and Scenedesmus sp. UKM9 biomass methanol extracts (ME) were prepared and tested against Gram-positive and Gram-negative bacteria. Chlorella sp. UKM8-ME showed the highest antibacterial activity. UKM8-ME minimum inhibitory concentrations were in the range of 0.312 to 6.25 mg/mL. Cytotoxicity evaluation using MTT assay showed that the microalgae methanolic extracts did not exhibit cytotoxicity against Vero-cells. The UKM8-ME was mainly containing 28 compounds from the Gas Chromatography-Mass Spectrometry (GC–MS) analysis. Major compounds of UKM8-ME included phenol (18.5%), hexadecanoic acid (18.25%), phytol (14.43%), 9,12-octadecadienoic acid (13.69%), and bicyclo[3.1.1]heptane (7.23%), which have been previously described to possess antimicrobial activity. Hence, Chlorella sp. (UKM8) methanol extracts showed promising antibacterial activity. More comprehensive studies are required to purify these antimicrobial compounds and develop our understanding on their mechanism in UKM8-ME to unleash their specific potential.     

Characterization and messenger activity of poly(A)-containing RNA from Chlorella.

Poly(A)-containing RNA was isolated by cellulose column chromatography from total RNA extracted from Chlorella fusca var. vacuolata 211/8p. RNA retained by the column was identified as poly(A)-containing RNA because it contained ribonuclease-resistant tracts, 25 to 55 nucleotides in length, from which not less than 80% of base was found to be adenine after acid hydrolysis. The base composition of poly(A)-containing RNA differed from that of RNA (largely ribosomal) which did not adsorb to cellulose, having a higher adenine content and a lower guanine content. Poly(A)-containing RNA was polydisperse including molecules with mobilities from 10S to 40S with a mean of about 20S. In an in vitro system derived from wheat-germ, protein synthesis was stimulated by adding poly(A)-containing RNA from Chlorella. Optimum conditions were established in this system with respect to the amount of poly(A)-containing RNA added and the concentration of KCl and Mg-2+. It is proposed that, in Chlorella, poly(A)-containing RNA includes cytoplasmic mRNA as has been shown for some other eucaryotic organisms.     

Identification and localization of a thylakoid-bound carbonic anhydrase from the green algae Tetraedron minimum (Chlorophyta) and Chlamydomonas noctigama (Chlorophyta)

In order to broaden our understanding of the eukaryotic CO₂-concentrating mechanism the occurrence and localization of a thylakoid-associated carbonic anhydrase (EC 4.2.1.1) were studied in the green algae Tetraedron minimum and Chlamydomonas noctigama. Both algae induce a CO₂-concentrating mechanism when grown under limiting CO₂ conditions. Using mass-spectrometric measurements of ¹⁸O exchange from doubly labelled CO₂, the presence of a thylakoid-associated carbonic anhydrase was confirmed for both species. From purified thylakoid membranes, photosystem I (PSI), photosystem II (PSII) and the light-harvesting complex of the photosynthetic apparatus were isolated by mild detergent gel. The protein fractions were identified by 77 K fluorescence spectroscopy and immunological studies. A polypeptide was found to immunoreact with an antibody raised against thylakoid carbonic anhydrase (CAH3) from Chlamydomonas reinhardtii. It was found that this polypeptide was mainly associated with PSII, although a certain proportion was also connected to light harvesting complex II. This was confirmed by activity measurements of carbonic anhydrase in isolated bands extracted from the mild detergent gel. The thylakoid carbonic anhydrase isolated from T. minimum had an isoelectric point between 5.4 and 4.8. Together the results are consistent with the hypothesis that thylakoid carbonic anhydrase resides within the lumen where it is associated with the PSII complex. Received: 13 May 2000 / Accepted: 16 August 2000     

Uncertainty in the determination of the molecular weight of poly(A)-containing RNA.

Estimates of the weight average molecular weight of mouse myeloma cell cytoplasmic poly(A)(+)RNA obtained by polyacrylamide-gel electrophoresis and by sucrose gradient sedimentation differ by a factor of two. This difference is not due to degradation of the RNA molecules during sedimentation nor to acid poly(A) double helix formation during electrophoresis. The difference is probably due, at least in part, to the presence of poly(A) sequences in the RNA molecules.     

Changes in the Levels of Ribulose-l,5-bisphosphate Carboxylase/Oxygenase (Rubisco) in Tetraedron minimum (Chlorophyta) during Light and Shade Adaptation

Exponentially growing cultures of the chlorophyta Tetraedronminimum were allowed to photoadapt to low (50µmole quantam^–2s^–1) and high (500µmole quanta m^–2–1)irradiance levels. In these cultures, various aspects of theorganization of the photosynthetic apparatus and related differencesin its performance were studied. In this organism, the observed five-fold increase in pigmentationof low-light adapted cells was due to increases in the numbersof PSU's, while their sizes remained constant. Using radioimmunoassay technique, we found that high-light adaptedalgae had over five times more Rubisco per PSU than their low-lightadapted counterparts. The high-light adapted algae also exhibited far higher (x2.3)light saturated photosynthetic rates per chl a. This increasewas the result of a reduction of tau, , the turnover time ofPS II reaction centers. We propose that the increase in Rubisco per PSU in high-lightadapted algae explains the reduction in , which results in thehigher P_max rates per chl a in these algae. The relationship is non linear, since the increase in Rubiscoper PSU was x5.3 whereas that in P_mM per chl a was only x2.3. (Received July 30, 1988; Accepted December 2, 1988)     

High molecular weight,cell surface-associated glycoprotein (fibronectin) lost in maglinant transformation

Fibronectin is a polymorphic glycoprotein found in blood and tissues of vertebrates and in cultures of adherent vertebrate cells. There are several forms of fibronectin is composed of two high molecular weight subunits held together by forms found in tissues and on and around the surfaces of cultured cells. Soluble fibronectin is composed of two high molecular weight subunits held together by disulfide bonds. Insoluble fibronectin may be covalently cross-linked in larger complexes. Fibronectin has affinities for collagen, fibrin, heparin, and cell surfaces. in culture, fibronectin in growth medium may mediate attachment of cells to substratum, and fibronectin synthesized by cells may mediate adhesion to substratum. The widespread occurrence of fibronectin in basal lamina indicates that many different cell types in vivo abut against a fibronectin-containing matrix. Cultured transformed cells usually lack cell-surface fibronectin, also called large, external transformation-sensitive (LETS) protein. The failure of transformed cells to synthesize or bind fibronectin is paralleled (at least in some systems) by failures to synthesize or bind collagen and proteoglycans. Abnormal synthesis of fibronectin and other matrix components and abnormal interactions with the tissue matrix may account for several phenotypic characteristics of transformed cultutred cells and for some of the malignant behavior of neoplastic cells in vivo.     

Immunoquantification of flavodoxin and ferredoxin from Scenedesmus vacuolatus (Chlorophyta) as iron-stress molecular markers

Quantification of the iron nutritional status of phytoplankton is of great interest not only for the study of the oceans but also for fresh waters. Flavodoxin is a small flavoprotein proposed as a molecular marker for iron stress, since it is induced as a consequence of iron deprivation, replacing the iron-sulphur protein ferredoxin. Flavodoxin and ferredoxin from Scenedesmus vacuolatus have been immunoquantified in cells grown under different iron nutritional conditions. Flavodoxin and ferredoxin levels correlate with the iron availability, and the calculated flavodoxin index can be used as an iron-stress marker. Other physiological parameters such as copper deficiency, heterotrophic or mixotrophic growth, nitrogen source and salt stress were also tested as potential factors influencing flavodoxin expression. Salt stress and heterotrophic growth conditions alter flavodoxin and ferredoxin expression. Once flavodoxin expression is repressed by iron (and severe deficiency alleviated), S.vacuolatus still increases its ferredoxin from 0·5 to 1·6 mol of ferredoxin per mole of ferredoxin-NADP⁺ reductase, and this ratio can be used for the evaluation of mild deficiency.     

High molecular weight DNA from nucleated erythrocytes for use in pulsed-field gel electrophoresis (PFGE)

Nucleated erythrocytes of lower vertebrates provide a source of genomic DNA that can be used in pulsed-field gel electrophoresis (PFGE) studies. Difficulties reported in the preparation of chicken erythrocyte DNA for analysis by PFGE suggest that the presence of hemoglobin iron may result in iron-mediated DNA degradation. We report here modifications to the procedures established for isolation of high molecular weight DNA from mammalian cells. By increasing the volume of buffers and extending the incubation periods to allow for the removal of hemoglobin iron, we have successfully prepared channel catfish erythrocyte DNA suitable for analysis of PFGE.     

High and low molecular weight proteolytic activities in prawn (Metapenaeus brevicornis) muscle

Muscle extract of prawn (Metapenaeus brevicornis) expressed high azocoll lytic activity compared to extracts of many other prawn varieties; the activity was also inhibited to a small extent by dithiothreitol. Ammonium sulphate precipitation, subsequent extraction at pH 5.6 and chromatography revealed the occurrence of two types of azocoll lytic activities: one, high molecular weight (630 kDa) and the other low molecular weight (< 30 kDa) enzyme. The former was stimulated by dithiothreitol whereas the latter was inhibited. SDS PAGE of high molecular weight preparation did not show homogeneity but the profile was similar to that of the low molecular weight fraction. Gel filtration of high molecular weight enzyme following incubation at high pH revealed the formation of low molecular weight fractions having activity towards azocoll. Chymotrypsin-like activity associated with high molecular weight enzyme was also susceptible to dissociation by high pH. Azocoll lytic activity of both enzymes was strongly inhibited by 1,10-phenanthroline.     

A chitin-like glycan in the cell wall of a Chlorella sp. (Chlorococcales,Chlorophyceae)

The stable amino-sugar fraction of the cell wall of the symbiotic Chlorella strain Pbi (Chlorophyceae) was isolated and investigated by sugar analysis, infra-red spectroscopy, lectin binding, enzymatic degradation, X-ray diffraction and electron microscopy. The results indicate the existence of a glycosaminoglycan which can be regarded as a chitin-like glycan. This carbohydrate structure is unusual for algae and reported here for the first time in unicellular chlorophycean algae.Abbreviations FITC fluorescein isothiocyanate - IR infra-red - NMR nuclear magnetic resonance - TFA trifluoroacetic acid - WGA wheat germ agglutinin We thank Peter Zugenmaier, Institut für Physikalische Chemie der Technischen Universität Clausthal-Zellerfeld, Germany, for valuable advice on X-ray diffraction techniques and for taking the Debye-Scherrer images. Wilfried Diekmann and David G. Robinson, Pflanzenphysiologisches Institut der Universität Göttingen, Germany, kindly carried out the freeze-etching. This work was supported by a fellowship from the Friedrich-Ebert-Stiftung to the first author and a grant from the Deutsche Forschungsgemeinschaft to the second author.     

紫芝细胞壁大分子量葡聚糖的纯化及结构鉴定

采用超微粉碎、热水浸提法从紫芝子实体水提残渣中获得细胞壁粗多糖,通过30%乙醇沉淀、20%醇洗的方法纯化出大分子量均一多糖GSCW30E-20E。苯酚硫酸法检测其多糖含量为98.03%,单糖组成分析显示其仅由葡萄糖组成,高效凝胶尺寸排阻色谱-多角度激光散射仪-示差折光检测技术测定其重均分子量为1.552×10 ⁶g/mol。通过红外光谱、甲基化及核磁共振分析对其结构进行解析,结果表明,GSCW30E-20E是一种β-D-葡聚糖,该多糖主链由β-(1,3)-糖苷键连接而成,每3个糖残基主链上通过β-(1,6)-糖苷键连有一个葡萄糖残基为支链。     

Interactions of the low molecular weight group of surfactant-associated proteins (SP 5-18) with pulmonary surfactant lipids

The interaction of the low molecular weight group of surfactant-associated proteins, SP 5-18, with the major phospholipids of pulmonary surfactant was studied by fluorescence measurements of liposomal permeability and fusion, morphological studies, and surface activity measurements. The ability of SP 5-18 to increase the permeability of large unilamellar lipid vesicles was enhanced by the presence of negatively charged phospholipid. The permeability of these vesicles increased as the protein concentration was raised and the pH was lowered. SP 5-18 also induced leakage from liposomes made both from a synthetic surfactant lipid mixture and from lipids separated from SP 5-18 during its purification from canine sources. When SP 5-18 was added to egg phosphatidylglycerol liposomes, the population of liposomes which became permeable leaked all encapsulated contents, while the remaining liposomes did not leak at all. The extent of leakage was higher in the presence of 3 mM calcium. SP 5-18 also induced lipid mixing between two populations of egg phosphatidylglycerol liposomes in the presence of 3 mM calcium, as monitored by resonance energy transfer between two different fluorescent lipid probes, N-(7-nitro-2,1,3-benzoxadiazol-4-yl)phosphatidylethanolamine and N-(lissamine rhodamine B sulfonyl)phosphatidylethanolamine. Negative-staining electron microscopy showed that the addition of SP 5-18 and 3 mM calcium produced vesicles twice the size of control egg phosphatidylglycerol liposomes. In addition, surface balance measurements revealed that the adsorption of liposomal lipids to an air/water interface was enhanced by the presence of SP 5-18, negatively charged phospholipids, and 3 mM calcium. These observations suggest a similar lipid dependence for the interactions observed in the fluorescence and adsorption experiments.     

Similarities in the molecular weight of poly(ADPR) polymerase from different tissues and species

Using the protein blotting technique, we determine the molecular weight of polyADPR polymerase from different tissues of beef, rat, chicken and pig directly in nuclear homogenates. We report here that the molecular weight of the enzyme is 130,000 in all samples tested.     

The Effect of a Quaternary Amine (Aliquat 336) on Growth and Photosynthesis of the Green Alga Chlorella emersonii, and the Effect on Photosynthesis in Isolated Spinach Chloroplasts

A quaternary amine, Aliquat 336, inhibits the growth of the green alga Chlorella emersonii, ¹⁴C-fixation of the alga is also inhibited. The effect and the site of action of the compound was studied by using isolated spinach chloroplasts. The carbon dioxide dependent oxygen evolution of the chloroplasts is inhibited directly upon the addition of the amine and the oxygen evolution is replaced by an oxygen uptake. By investigating some electron transport reactions in the chloroplasts we were able to show that Aliquat 336 affects the electron transport on the level of photophosphorylation. The results from the in vivo and the in vitro experiments thus show that the quaternary amine affects the photosynthetic process. Aliquat 336 is a solvent extractant used in several industrial processes for extraction of metals from aqueous solutions. Aliquat 336 could be considered a presumptive water pollutant as the compound could enter a recipient water body and thus affect photosynthesis.     

Relationships among the subunits of the high molecular weight proteinase,macropain (proteasome)

An analysis of the subunits of the high molecular weight proteinase, macropain (multicatalytic proteinase or proteasome) from human erythrocytes has been conducted using N-terminal amino acid sequencing, gel electrophoresis and reverse-phase peptide mapping. This analysis provided evidence for the existence of 13 subunits of different primary structure. Five subunits were susceptible to the Edman degradation and yielded unique N-terminal sequences. Similarities among these sequences, however, indicated that the subunits are homologues. Two-dimensional gel electrophoresis discriminated 10 major components, which included two of the subunits for which N-terminal sequences had been determined and eight N-terminally blocked subunits. Tryptic peptide mapping indicated that all 10 of these components have a different amino acid sequence. Tryptic peptides from some of the subunits were subjected to amino acid sequence analysis, and the data indicated that all the subunits tested in this way are related by common ancestry. The data suggest that at least nine of the total of 13 subunits are encoded by members of the same gene family; the remaining four subunits have not yet been investigated in sufficient detail to establish their relationships. No evidence for a close relationship with any previously investigated proteinase family has been found. Finally, through a comparison of the ‘latent’ and ‘active’ forms of macropain, the study established a close similarity in the subunit composition of these catalytically very different species, although proteolytic degradation of selected subunits appears in the active form of the enzyme.     

Molluscan death effector domain (DED)-containing caspase-8 gene from disk abalone (Haliotis discus discus): molecular characterization and expression analysis

The caspase family represents aspartate-specific cysteine proteases that play key roles in apoptosis and immune signaling. In this study, we cloned the first death effector domain (DED)-containing molluscan caspase-8 gene from disk abalone (Haliotis discus discus), which is named as hdCaspase-8. The full-length hdCaspase was 2855 bp, with a 1908 bp open reading frame encoding 636 amino acids. The hdCaspase-8 had 72 kDa predicted molecular mass with an estimated isoelectric point (PI) of 6.0. The hdCaspase-8 amino acid sequence contained the characteristic feature of an N-terminal two DED, a C-terminal catalytic domain and the caspase family cysteine active site ⁵¹³KPKLFFLQACQG⁵²⁴. Phylogenetic analysis results showed that hdCaspase-8 is more similar to the invertebrate Tubifex tubifex (sludge worm) caspase-8.Real-time RT-PCR results showed that hdCaspase-8 constitutively and ubiquitously expressed in all tested tissue of unchallenged disk abalone. The basal expression level of hdCaspase-8 in gill tissue was higher than all other tested tissues. The hdCaspase-8 mRNA expression in gill and hemocytes was significantly up-regulated by exposure to bacteria (Vibrio alginolyticus, Vibrio parahemolyticus and Listeria monocytogenes) and VHSV (viral hemorrhagic septicemia virus), as compared to control animals. These results suggest that hdCaspase-8 may be involved in immune response reactions in disk abalone.     

Purification and characterization of a high molecular weight proteinase (macropain) from human erythrocytes

An alkaline proteinase, previously identified in rat liver and heart, has been purified from the soluble fraction of human erythrocytes. The proteinase has an apparent molecular weight of 600 000 and is composed of eight subunits with molecular weights ranging from 32 000 to 21 000. The proteinase degrades both protein and synthetic peptide substrates with a broad pH optimum of 7.5-11.0. Among the synthetic peptides tested, tripeptides with arginine at the P1 position (e.g. Z-Val-Leu-Arg-4-methoxy-2-napthylamine and Boc-Leu-Gly-Arg-4-methylcoumarin-7-amide) are particularly good substrates. The proteinase appears to be sulfhydryl-dependent and is inhibited completely by mersalyl acid and by hemin; inhibitors of serine and metallo-type proteinases have no effect on proteinase activity. Interestingly, a variety of other proteinase inhibitors such as leupeptin, chymostatin and N-ethylmaleimide failed to completely inhibit protein-hydrolyzing activities of the enzyme. These results indicate that these activities may be accounted for by at least two different catalytic sites. Proteinase activity is stable in the presence of 1 M urea, 0.5% Triton X-100 or 0.03% SDS and is not affected by ATP. Based on the high molecular weight and sulfhydryl-dependence, we have named this proteinase macropain.