NDK Interacts with FtsZ and Converts GDP to GTP to Trigger FtsZ Polymerisation - A Novel Role for NDK

Introduction

Nucleoside diphosphate kinase (NDK), conserved across bacteria to humans, synthesises NTP from NDP and ATP. The eukaryotic homologue, the NDPK, uses ATP to phosphorylate the tubulin-bound GDP to GTP for tubulin polymerisation. The bacterial cytokinetic protein FtsZ, which is the tubulin homologue, also uses GTP for polymerisation. Therefore, we examined whether NDK can interact with FtsZ to convert FtsZ-bound GDP and/or free GDP to GTP to trigger FtsZ polymerisation.

Methods

Recombinant and native NDK and FtsZ proteins of Mycobacterium smegmatis and Mycobacterium tuberculosis were used as the experimental samples. FtsZ polymersation was monitored using 90° light scattering and FtsZ polymer pelleting assays. The γ³²P-GTP synthesised by NDK from GDP and γ³²P-ATP was detected using thin layer chromatography and quantitated using phosphorimager. The FtsZ bound ³²P-GTP was quantitated using phosphorimager, after UV-crosslinking, followed by SDS-PAGE. The NDK-FtsZ interaction was determined using Ni²⁺-NTA-pulldown assay and co-immunoprecipitation of the recombinant and native proteins in vitro and ex vivo, respectively.

Results

NDK triggered instantaneous polymerisation of GDP-precharged recombinant FtsZ in the presence of ATP, similar to the polymerisation of recombinant FtsZ (not GDP-precharged) upon the direct addition of GTP. Similarly, NDK triggered polymerisation of recombinant FtsZ (not GDP-precharged) in the presence of free GDP and ATP as well. Mutant NDK, partially deficient in GTP synthesis from ATP and GDP, triggered low level of polymerisation of MsFtsZ, but not of MtFtsZ. As characteristic of NDK’s NTP substrate non-specificity, it used CTP, TTP, and UTP also to convert GDP to GTP, to trigger FtsZ polymerisation. The NDK of one mycobacterial species could trigger the polymerisation of the FtsZ of another mycobacterial species. Both the recombinant and the native NDK and FtsZ showed interaction with each other in vitro and ex vivo, alluding to the possibility of direct phosphorylation of FtsZ-bound GDP by NDK.

Conclusion

Irrespective of the bacterial species, NDK interacts with FtsZ in vitro and ex vivo and, through the synthesis of GTP from FtsZ-bound GDP and/or free GDP, and ATP (CTP/TTP/UTP), triggers FtsZ polymerisation. The possible biological context of this novel activity of NDK is presented.     

Activation of halophilic nucleoside diphosphate kinase by a non-ionic osmolyte,trimethylamine N-oxide

The folding and activity of halophilic enzymes are believed to require the presence of salts at high concentrations. When the inactivated nucleoside diphosphate kinase (NDK) from extremely halophilic archaea was incubated with low salt media, no activity was regained over the course of 8 days. When it was incubated with 2 M NaCl or 3 M KCl, however, it gradually regained activity. To our surprise, trimethylamine N-oxide (TMAO) also was able to induce activation at 4.0 M. The enzyme activity and secondary structure of refolded NDK in 4 M TMAO were comparable with those of the native NDK or the refolded NDK in 3.8 M NaCl. TMAO is not an electrolyte, meaning that the presence of concentrated salts is not an absolute requirement, and that charge shielding or ion binding is not a sole factor for the folding and activation of NDK. Although both NaCl and TMAO are effective in refolding NDK, the mechanism of their actions appears to be different: the effect of protein concentration and pH on refolding is qualitatively different between these two, and at pH 8.0 NDK could be refolded in the presence of 4 M TMAO only when low concentrations of NaCl are included.     

Degradation of chloroplast DNA in second leaves of rice (Oryza sativa) before leaf yellowing

Summary The second leaf ofOryza sativa develops, grows and ages within the 10 days that follow imbibition under our controlled continuous-light conditions. Proplastids in the leaf cells develop, mature to become chloroplasts and then age and disintegrate. In an examination of this life process, we studied first the behavior and the number of copies of plastid DNA and levels of chlorophyll by epifluorescence microscopy after staining with 4,6-diamidino-2-phenylindole (DAPI), and by fluorimetry with a video-intensified microscope photon-counting system (VIMPCS). The results indicated that the number of copies of the plastid DNA per plastid increased and reached to plateau value of approximately 100 at the time when the elongation of the mesophyll cells and the enlargement of chloroplasts ceased 96 h after imbibition. However, 24 h later, the number of copies of plastid DNA per chloroplast began to decrease and fell rapidly to approximately 30 copies within 168 h after imbibition. Our examination of the number of chloroplasts per mesophyll cell indicated that no division of chloroplasts occurred more than 72 h after imbibition. The results suggest that the decrease in number of copies of plastid DNA per chloroplast was not due to an increase in the number of chloroplasts, but that this decrease was caused by degradation by unidentified enzymes. Since visible senescence of leaves, which was characterized by development of a yellowish color, began 168 h after imbibition, the degradation of plastid DNA seemed to occur 48 h before the visible leaf senescence. When we tested the nucleolytic activities in the second leaves after imbibition by digestion of plasmids in vitro and DNA-SDS polyacrylamide gel electrophoresis, five Ca²⁺–, four Zn²⁺–, and four Mn²⁺–dependent nucleases were detected in the leaf blades, and one of the Ca²⁺–, two of the Zn²⁺–, and two of the Mn²⁺–dependent nucleases were also identified in a purified preparation of intact chloroplasts. When the activity of the Zn²⁺–dependent nucleases (51 kDa and 13 kDa) increased markedly, degradation of the plastid DNA occurred. These results suggest that the destruction of chloroplast DNA, which occurs approximately 48 h before leaf yellowing, could be due to the activation of some metallo-nucleases and, furthermore, this enzymatic degradation propels the leaf towards senescence.     

BiP, HSP70, NDK and PDI in wheat endosperm. II. Effects of high temperature on protein and mRNA accumulation

The effects of high temperature on accumulation of the 70‐kDa heat shock protein (HSP70) and nucleoside diphosphate kinase (NDK) as well as two other proteins that have roles in the biosynthesis of storage proteins were examined during grain development. An HSP70 homolog and a 17‐kDa NDK were co‐purified from wheat endosperm, their identity verified, and a cDNA for an HSP70 expressed in endosperm was isolated. Wheat plants ( Triticum aestivum , cvs Butte and Vulcan) were heat shocked at 40°C or exposed to maximum daily temperatures of 37 or 40°C during early or mid‐grain fill. Antibodies and cDNA probes for BiP, HSP70, NDK and PDI were used to examine the effect of high temperatures on the accumulation of protein and mRNA in the endosperm. HSP70 mRNA levels increased substantially when plants were exposed to heat shock or to a 1‐day gradual increase to 40°C. The effects of a 5‐day heat treatment on mRNA levels were more complicated and depended on the developmental stage of the grain. A treatment that began at 7 days post‐anthesis (DPA) decreased the level of mRNA for HSP70, BiP, PDI and NDK, whereas a treatment that began at 14 DPA slightly increased mRNA levels. The same treatments increased the accumulation of HSP70 but did not affect BiP, PDI, or NDK protein levels. This is the first detailed report on the effects of heat on mRNA and protein levels for HSP70 in a developing seed storage tissue.     

Uptake of auxotrophic cells of a heterocyst-forming cyanobacterium by tobacco protoplasts,and the fate of their associations

During imbibition, exogenous myo-inositol (MI) was readily introduced into the free MI pool of germinating wheat (Triticum aestivum L.). Maximum uptake, 70 g per caryopsis or 1.5 mg g^–1 of caryopsis, was reached at 0.05 M MI. Movement of free MI within the germinating caryopsis was traced with [2-³H]MI by two procedures, uptake by imbibition and injection into softened endosperm. The former procedure was useful during initial stages of germination; the latter provided a means of tracing the metabolic fate of MI generated by hydrolysis of phytate during mobilization of reserves within the caryopsis. In both procedures, the bulk of the added label was transferred to the seedling where it appeared in uronosyl and pentosyl units of 80% ethanol-insoluble polysaccharides, 2-O, C-Methylene-MI, an inhibitor of the MI oxidation pathway, blocked the utilization of [2-³H]MI as well as d-[1¹⁴C]glucose for biogenesis of pentose-and uronic-acid-containing polysaccharides.Abbreviations MI myo-inositol - OCM-MI 2-O, C-methylene-myo-inositol     

Protein utilization during starvation in fat and lean Svalbard ptarmigan (Lagopus mutus hyperboreus)

Summary Body protein sparing during starvation has been examined in fat and lean Svalbard ptarmigan. Protein utilization was determined from daily N excretion and from the rate of decrease in body mass. Changes in plasma concentrations of -hydroxybutyrate, free fatty acids, glucose, and uric acid were also recorded. When fat birds were starved for 15 days protein catabolism initially fell (phase I) and was thereafter kept low (phase II). This was evident from the temporal pattern in both N excretion and body mass loss. In two birds, N excretion eventually increased, revealing enhanced protein catabolism and thus a third phase of starvation. Changes in protein utilization were paralleled by changes in plasma uric acid. Approximately 9% of the energy demand was covered by breakdown of body protein during phase II. The importance of fat catabolism in providing energy was indicated by markedly elevated plasma levels of -hydroxybutyrate and free fatty acids. When lean birds were starved for 5 days there appeared to be no phase II. The temporal pattern of body mass loss indicated phase I and III but that of N excretion only phase III. The relative contribution of body protein to energy demand increased from 22% at day 2 to 41% at the end of starvation and was paralleled by increased plasma uric acid. When data from lean and fat birds were pooled, the changes in uric acid and N excretion were highly correlated (r=0.92, P<0.001), indicating that plasma uric acid is a reliable index of protein breakdown in starving Svalbard ptarmigan. In conclusion, starving fat Svalbard ptarmigan have a much greater capacity to spare body protein than lean birds. Fat birds effectively reduce protein catabolism and maintain this at a low level whereas starving lean birds increase protein catabolism.Abbreviations -OHB -hydroxybutyrate - BM body mass - BMR basal metabolic rate; dne daily nitrogen excretion - FFA free fatty acids - MR metabolic rate     

Molecular cloning and nucleotide sequence cDNA encoding nucleoside diphosphate kinase of rice (Oryza sativa L.)

We isolated a rice cDNA encoding nucleoside diphosphate kinase (NDK, EC 2.7.4.6). The deduced amino acid sequence of the rice NDK shows highest homology to spinach NDK-I. The rice NDK gene exhibits a strong codon bias (73.8% GC) in the third position of the codon. DNA blot analysis indicated that at least single NDK gene is present in rice genome.     

Characterization of the 3' --> 5' exonuclease activity found in human nucleoside diphosphate kinase 1 (NDK1) and several of its homologues

Nucleoside diphosphate kinases (NDKs), an evolutionarily conserved family of proteins, synthesize nucleoside triphosphates from nucleoside diphosphates and ATP. Here, we have characterized the kinase activity and DNA processing functions of eight human proteins that contain at least one domain homologous to Escherichia coli NDK. Not all human proteins with NDK-like domains exhibited NDK activity when expressed as recombinant proteins in E. coli. Human NDK1 (NM23-H1) has been reported to have 3' --> 5' exonuclease activity. In addition to human NDK1, we also find that human NDK5, NDK7, and NDK8 contain 3' --> 5' exonuclease activity. Site-directed mutagenesis, competition assays between wild-type and mutant NDK proteins, and NMR studies confirmed that the DNA-binding and 3' --> 5' exonuclease activity of human NDK1 is an intrinsic activity of the protein. Using double-stranded DNA substrates containing modified bases, human NDK1 efficiently excised nucleotides from the single-strand break produced by APE1 or Nth1. When human cells were treated with various DNA-damaging agents, human NDK1 translocated from the cytoplasm to the nucleus. These results suggest that, in addition to maintenance of nucleotide pool balance, the human NDK-like proteins may have previously unrecognized roles in DNA nucleolytic processing.     

Preferential digestion of chloroplast nuclei (nucleoids) during senescence of the coleoptile ofOryza sativa

Summary The coleoptile ofOryza sativa develops, grows and ages within 4 days that follow imbibition. It is, thus, a very useful system for experimental analysis of the life cycle of organelles, for example, the development, growth and aging of plastids in higher plants. We examined the behavior and levels of DNA and chlorophyll in the plastid by epifluorescence microscopy after staining with 4-6-diamidino-2-phenylindole (DAPI), and by fluorimetry with a video-intensified-photon counting system (VIMPCS). The whitish yellow coleoptile appeared soon after imbibition and, between the first 24 and 60 h that followed imbibition, it grew markedly in a longitudinal direction, with concomitant elongation of the cells, and an increase in the volume of plastids and in the amount of DNA in the plastids. The chlorophyll content per plastid began to increase when the coleoptile turned green, 48 h after imbibition, and reached a plateau value when the coleoptile was 3.5 mm in length, 72 h after imbibition. More than 12 h later, the chlorophyll disappeared just before the breakdown of chloroplasts was initiated. Proplastids in young coleoptiles, contained a plastid nucleus which was located in the central area of the plastids and each nucleus consisted of approximately 6 copies of plastid DNA (ptDNA). The number of copies of ptDNA per plastid increased gradually, with a concomitant increase in the volume of the plastids after imbibition, and reached approximately 130 times the value in the young proplastids, 60 h after imbibition, when the plastid developed into a chloroplast. However, each plastid nucleus did not scatter throughout the entire interior region of each chloroplast. The disappearance of each plastid nucleus occurred more than 12 h before the degeneration of the chloroplasts. The number of plastids per cell increased from 10 to 15 in young coleoptiles within 12 h after imbibition. Yet the number remained constant throughout subsequent growth and aging of the coleoptile. Thus the preferential reduction in the amount of chloroplast DNA was not due to the division of the plastid but could, perhaps, be associated directly with the aging of the cells of the coleoptile which precedes senescence of the coleoptiles.     

Interspecific hybridization of cultivated rice, Oryza sativa L. with the wild rice, O. minuta Presl.

Crosses were made between four varieties (Mahsuri, Setanjung, MR84 and MR103) of Oryza sativa L. (2n=24, AA) and one accession of O. minuta (2n= 8, BBCC). The seed set obtained ranged between 9.5% and 25.1% depending on the rice variety used. By rescuing 14-day-old embryos and culturing them on 25%-strength MS medium we obtained a total of 414 F₁ hybrids. The F₁s were vigorous, tillered profusely, were perennial and male-sterile. The hybrids were triploid (ABC) with 36 chromosomes and showed irregular meiosis. The average frequency and range of chromosome associations at metaphase I or early anaphase I pollen mother cells of F₁ plants were 29.31(16–36) Is +3.32(0–10) IIs+0.016(0–1) IIIs+0.002(0–1) IVs. Upon backcrossing the original triploid hybrids and colchicine-treated hybrids to their respective recurrent parents, and further embryo rescue, 17 backcross-1 (BC₁) plants were obtained. Of all the crosses using MR84, no BC₁ plant was obtained even after pollinating 13 894 spikelets of the triploid hybrid. The BC₁s were similar in appearence to the F₁s and were male-sterile, their chromosome number ranged from 44 to 48. By backcrossing these BC₁s and nurturing them through embryo rescue, we obtained 32 BC₂ plants. Of these, however, only 18 plants grew vigorously. One of these plants has 24 chromosomes and the other 17 have chromosome numbers ranging between 30 and 37. The 24-chromosome plant was morphologically similar to the O. sativa parent and was partially fertile with a pollen and spikelet fertility of 58.8% and 12.5% respectively. All of the F₁ and BC₁ plants were found to be resistant to five Malaysian isolates (XO66, XO99, XO100, XO257 and XO319) of Xanthomonas campestris pv oryzae. Amongst the BC₂s, the reaction varied from resistant to moderately susceptible. The 24-chromosome BC₂ plant was resistant to the four isolates and moderately resistant to isolate XO100 to which the O. sativa parent was susceptible.Part of PhD thesis submitted by first author to Universiti Kebangsaan Malaysia, Bangi     

The endoplasmic reticulum of mung-bean cotyledons: Quantitative morphology of cisternal and tubular ER during seedling growth

The ultrastructure of the endoplasmic reticulum (ER) in storage parenchyma cells in the cotyledons of mung beans (Vigna radiata L.) was examined during germination and seedling growth. Two different methods were used to visualize the ER: thin (0.08 m) sections of tissue fixed in formaldehyde and glutaraldehyde and post-fixed with osmium tetroxide, and thick (1 m) sections of tissue fixed in buffered aldehyde and post-fixed with zinc iodide-osmium tetroxide (ZIO). Changes in relative amounts of ER were quantified by morphometry (stereology).The ER occurs in two forms: a cisternal form with associated ribosomes which can be seen at all stages from imbibition to cotyledon senescence, and a tubular form which initially has associated ribosomes. Stereoscopic images of thick sections of cotyledons of 2-day-old seedlings show that the tubular ER consists of a three-dimensional array of interconnecting tubules which have numerous connections with the cisternal ER. The network of tubules and cisternae extends throughout the cytoplasm enveloping the protein bodies. Germination and seedling growth are accompanied by a reduction in the total volume occupied by the ER. This reduction is the result of a preferential loss of tubular ER and occurs largely before protein mobilization. Cisternal ER decreases during the first 48 h of imbibition and seedling growth, but storage cells subsequently show an increase in cisternal ER just prior to and during the period of protein mobilization. Cisternal ER remains conspicuous during the last phase of reserve mobilization when starch is broken down and the cells are starting autophagy.Abbreviations ER endoplasmic reticulum - ZIO zinc iodide-osmium tetroxide This is the second in a series of papers on the endoplasmic reticulum of mung bean cotyledons. The first paper is referenced herein as Gilkes and Chrispeels (1980)     

Crystal structure of nucleoside diphosphate kinase required for coleoptile elongation in rice (Oryza sativa L.)

Nucleoside diphosphate kinase (NDK) is a ubiquitous enzyme found in all organisms and cell types, and catalyzes the transfer of the phosphoryl group from a nucleoside triphosphate to a nucleoside diphosphate. The enzyme is involved in and required for coleoptile elongation in rice as the level of the rice NDK (rNDK) changes during seed germination and the early stages of seedling growth. The expression of rice NDK gene is up-regulated in the growing coleoptiles when the anaerobic stress persists. The rNDK structure determined at 2.5 A resolution consists of a four-stranded anti-parallel beta-sheet, of which the surfaces are partially covered with six alpha-helices; its overall and active site structures are similar to those of homologous enzymes except the major conformation variations of residue 132-138 regions, involving significant structural contacts. The model contains 148 residues of 149 residues in total and averaged 19 water molecules per monomer for 12 molecules in an asymmetric unit. A mold of 12 superimposed molecules shows that the alphaA-alpha2 area has greater variations and higher temperature factors, indicating the flexibility for a substrate entrance. Hexameric molecular packing in both crystal and solution implies that rNDK functions as hexamers. This rNDK structure, which is the first NDK structure from a higher plant system, provides the structural information essential to understand the functional significance of this enzyme during growth and development in both rice and other plants.     

Reversible phosphorylation of tonoplast proteins involves tonoplast-bound calcium-calmodulin-dependent protein kinase(s) and protein phosphatase(s)

In highly purified tonoplast fractions from Acer pseudoplatanus cells, the in vitro reversible phosphorylation of proteins affected only a restricted set of polypeptides. The phosphorylation process has been shown to be dramatically stimulated by calcium via the mediation of calmodulin as the transducer. The protein kinase(s) was totally inhibited by micromolar concentrations of a calmodulin antagonist. Tonoplast appears to be potentially a good experimental system for the evaluation of the effects of protein phosphorylation on membrane properties in plants.Abbreviations CaM calmodulin - EGTA ethylene glycol-bis-(-amino ethylether)N,N,NN-tetraacetate - SDS-PAGE sodium dodecylsulphate polyacrylamide gel electrophoresis - TCA trichloroacetic acid     

Molecular cloning,sequence determination and heterologous expression of nucleoside diphosphate kinase from Pisum sativum

Protein sequence data derived from the N-terminal region of a 17 kDa polypeptide associated with the microsomal membrane fraction from Pisum sativum was used to design degenerate oligonucleotides which were used to amplify P. sativum cDNA via the polymerase chain reaction (PCR). Amplified cDNA was used as a probe to screen a P. sativum cDNA library and a cDNA clone, NDK-P1 was isolated and sequenced. The protein encoded by NDK-P1 had a calculated molecular mass of 16485 Da and possessed substantial homology with nucleoside diphosphate kinases (NDKs) isolated and cloned from other sources. High levels of expression of NDK-P1 protein were achieved in Escherichia coli using a T7-driven expression system. Recombinant NDK-P1 protein was shown to possess NDK activity and had similar biochemical characteristics to NDKs isolated from other sources. The Michaelis constants for a variety of nucleoside diphosphate (NDP) substrates were found to be broadly similar to those reported for other NDKs, with thymidine nucleotides being the sustrates of greatest affinity.     

Flavonoid genetics of the 28-chromosome “Siberian” Iris

Summary A Chromatographic survey of flavonoids in the various flower color mutants of the 28-chromosome Siberian Iris (series Sibiricae subseries Sibiricae) was conducted using mutants of known genotype (Vaughn 1974). Mutants at the C locus contain the malvidin glycoside ensatin, indicating that this gene locus may control methylation of delphinidin. Clear white, a mutation at the W locus, results in the production of flavones in excess.     

Litterfall,leaf decomposition and litter colonization of Tessaria integrifolia (compositae) in the Parana river floodplain

Litterfall and leaf decomposition rates were measured in Choui Island, 45 km downstream from the confluence of the Paraná and Paraguay rivers. The material was collected biweekly from April 1985 through September 1986. Decomposition was measured in situ by the litter bag technique.Annual litterfall of Tessaria integrifolia gallery forest measured in the period April 1985 to March 1986 was 8.15 t ha^-1. Leaf litterfall was seasonal, i.e. significantly less leaf litter was shed during the high water phase than during the low water phase. The half life of the T. integrifolia litter over 38 days of decomposition was 20 days. At the beginning of the experiment, 15 and 38 days subsamples of remaining detritus were analyzed in order to determine changes in the nutrient content. After 38 days of incubation, the order of nutrient disappearance was Ca > K > N > Mg > Na > P.The number of invertebrates per g remaining litter of Tessaria integrifolia increased between incubations days 7 and 31. Collector-gatherers were more abundant after 38 days incubation; there were no shredders colonizing the leaf litter bags.     

Identification of an ascorbate-dependent cytochrome<Emphasis Type="Italic"> b</Emphasis> of the tonoplast membrane sharing biochemical features with members of the cytochrome<Emphasis Type="Italic"> b</Emphasis>561 family

Two membrane-bound, ascorbate-dependent b-type cytochromes were identified in etiolated bean (Phaseolus vulgaris L.) hypocotyls. Following solubilization of microsomal membranes and anion-exchange chromatography at pH 8.0, two major cytochrome peaks (P-I and P-II) were separated. Both cytochromes were reduced by ascorbate and re-oxidized by monodehydroascorbate, but P-I reduction by ascorbate was higher and saturated at far lower concentrations of ascorbate with respect to P-II. The -band was symmetrically centered at 561 nm in P-I, but it was asymmetric in P-II with a maximum at 562 nm and shoulder at 557 nm. Ascorbate reduction of P-II, but not P-I, was inhibited by diethyl pyrocarbonate. Reduced P-II but not P-I was readily oxidized by certain ferric chelates, including FeEDTA and Fe-nitrilotriacetic acid. Purified P-I, associated with the plasma membrane, showed up as a 63-kDa glycosylated protein during sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) and behaved as a monomer of about 70 kDa during size-exclusion chromatography. P-I identified with a previously purified ascorbate-dependent b-type cytochrome of bean hypocotyl plasma membranes [P. Trost et al. (2000) Biochim Biophys Acta 1468:1–5]. Partially purified P-II, on the other hand, correlated with a heme-protein of 27 kDa in SDS–PAGE gels, was dimeric (60 kDa) during size-exclusion chromatography, and was associated with the tonoplast marker V-ATPase in sucrose gradients. The sequence of a peptide of 11 residues obtained by tryptic digestion of P-II was found to be identical to a segment of a putative cytochrome b561 of Zea mays and highly conserved in other related plant sequences, including that of Arabidopsis thaliana cytochrome b561-1 (CAA18169). The biochemical features fully support the assignment of P-II cytochrome to the family of cytochrome b561, ascorbate-dependent (CYBASC) cytochromes, which also includes cytochrome b561 of animal chromaffin granules. The presence of a cytochrome reducing ferric chelates on the tonoplast is consistent with the role of plant vacuoles in iron homeostasis.     

Growth ofEucheuma isiforme (C. Agardh) J. Agardh on experimental rafts off the coast of Yucatan State,Mexico

A field experiment was carried out at two sites off Yucatan State, Southeast Mexico, in order to determine the feasibility of culturing the red seaweedEucheuma isiforme; this was done during May–September 1989. At both sites (Uaymitun and Dzilam) the 25 days harvest and 14 algae per line plant density growth rates (2.21% day^–1 and 1.21% day^–1, respectively) were significantly higher (p<0.05) than those obtained with other combinations of the two factors tested (50, 75, 100 and 125 days harvest and 9 and 14 algae per line plant density). The mean carrageenan content of the cultured algae was 35.8% and 31.4% at Uaymitun and Dzilam, respectively.     

Preliminary investigations on the utilization of tubificid worms by postlarvae of Macrobrachium lanchesteri (de Man)

Postlarvae of Macrobrachium lanchesteri were stocked in confined waters. The larvae were fed on an ad libitum food of Tubifex tubifex worms for 30 days and their rearing has been established. 1 Kg of initially stocked postlarvae need 4.5 Kg of live T. tubifex to produce 0.537 Kg of new flesh. The worms were found to increase the crude protein content of larval flesh by 17% of the dry matter.     

Host and bacterial factors that regulate LC3 recruitment to Listeria monocytogenes during the early stages of macrophage infection

Listeria monocytogenes is a bacterial pathogen that can escape the phagosome and replicate in the cytosol of host cells during infection. We previously observed that a population (up to 35%) of L. monocytogenes strain 10403S colocalize with the macroautophagy marker LC3 at 1 h postinfection. This is thought to give rise to spacious Listeria-containing phagosomes (SLAPs), a membrane-bound compartment harboring slow-growing bacteria that is associated with persistent infection. Here, we examined the host and bacterial factors that mediate LC3 recruitment to bacteria at 1 h postinfection. At this early time point, LC3⁺ bacteria were present within single-membrane phagosomes that are LAMP1⁺. Protein ubiquitination is known to play a role in targeting cytosolic L. monocytogenes to macroautophagy. However, we found that neither protein ubiquitination nor the ubiquitin-binding adaptor SQSTM1/p62 are associated with LC3⁺ bacteria at 1 h postinfection. Reactive oxygen species (ROS) production by the CYBB/NOX2 NADPH oxidase was also required for LC3 recruitment to bacteria at 1 h postinfection and for subsequent SLAP formation. Diacylglycerol is an upstream activator of the CYBB/NOX2 NADPH oxidase, and its production by both bacterial and host phospholipases was required for LC3 recruitment to bacteria. Our data suggest that the LC3-associated phagocytosis (LAP) pathway, which is distinct from macroautophagy, targets L. monocytogenes during the early stage of infection within host macrophages and allows establishment of an intracellular niche (SLAPs) associated with persistent infection.