Particulate and Dissolved Organic Carbon Production by the Heterotrophic Nanoflagellate Pteridomonas danica Patterson and Fenchel

Abstract We established a budget of organic carbon utilization of a starved heterotrophic nanoflagellate, Pteridomonas danica, incubated in batch cultures with Escherichia coli as model prey. The cultures were sampled periodically for biomass determinations and total organic carbon dynamics: total organic carbon, total organic carbon <1 μm, and dissolved organic carbon (DOC, <0.2 μm). During the 22 h incubation period, P. danica underwent biovolume variations of 3.2-fold. Gross growth efficiency was 22% and net growth efficiency 40%. P. danica respired 33% and egested 44% of the ingested E. coli carbon during lag and exponential growth phases. The form of the organic carbon egested varied. Of the total ingested carbon, 9% was egested in the form of DOC and occurred mainly during the exponential growth phase; 35% was egested in the form of particulate organic carbon (POC), ranging in size from 0.2 to 1 μm, and took place during the lag phase. P. danica could have reingested as much of 58% of this previously produced POC during the exponential growth phase as food scarcity increased. We concluded that POC egestion by flagellates could represent a significant source of submicrometric particles and colloidal organic matter. In addition, flagellate reingestion of egested POC could play a nonnegligible role in the microbial food web. Finally, the methodology reported in this study has proved to be a useful tool in the study of carbon metabolism in aquatic microorganisms. Received: 31 July 1998; Accepted: 2 March 1999     

Phagocytosis reveals a reversible differentiated state early in the development of the mouse embryo

Mural trophectoderm cells of the mouse embryo possess a phagocytic potential as early as 3.5 days post coitum (d.p.c.). This first differentiated function shows a graded variation along the embryonic-abembryonic axis, from a maximal activity in the non-dividing cells of the abembryonic pole to a complete lack of activity in the replicating polar trophectoderm overlying the inner cell mass (ICM). This pattern can be explained by a negative control exerted by the ICM. Addition of FGF4, a factor secreted by ICM cells, strongly inhibited phagocytosis while inducing resumption of DNA synthesis in mural trophectoderm cells, revealing a reversible, FGF4-dependent differentiation state. Under conditions in which a small cluster of mural trophectoderm cells (<10) had internalized large particles, these otherwise morphologically normal embryos could not implant in the uterus, indicating that cells at the abembryonic pole have a critical role in initiating the implantation process. At post-implantation stages (6.5-8.5 d.p.c.), the ectoplacental cone and secondary giant cells derived from the polar trophectoderm also contained active phagocytes, but at that stage, differentiation was not reversed by FGF4.     

Polar lipid biomarkers of free-living bacteria from oligotrophic marine waters

Free-living marine bacteria isolated from oligotrophic Mediterranean waters were enriched in culture to characterize their phospholipid fatty acids (PLFAs). Odd chain iso- and anteiso-FAMEs and n–16:0 were the predominant structural PLFAs, together with a homologous series identified as mid-chain methoxy FAMEs. The dominant methoxy fatty acids identified were 9-CH₃O-15:0, 9-CH₃-16:0 and 11-CH₃O-17:0, occuring as pairs of stereoimers. Methoxy fatty acids accounted for up to 37% of PLFAs of free-living bacteria, which sets them as promising new biomarkers for bacteria of oligotrophic waters. Although similar homologues have already been characterized in a variety of eukaryotes and prokaryotes, methoxy fatty acids are identified here for the first time in marine bacteria. Analytical difficulties that may hinder the characterization of these biomarkers are presented, and structural elucidation keys by gas chromotography coupled to mass spectrometry are discussed. Whilst bacterial branched fatty acids were transferred to storage lipids of bacterivorous flagellates methoxy acids were not transferred to higher trophic levels in the studied conditions.     

Caspase-14 but not caspase-3 is processed during the development of fetal mouse epidermis

The activation of caspases is a central step in apoptosis and may also be critical for terminal differentiation of epidermal keratinocytes (KC). In particular, caspase-3 has been implicated in the differentiation of embryonic KC as well as in programmed cell death of KC, and caspase-14 has been suggested to function in the formation or homeostasis of the stratum corneum (SC). To test the putative roles of these proteases, we determined their expression level and activation status during development of fetal mouse epidermis. The level of procaspase-3 did not change significantly during epidermal development, and enzyme activation was undetectable at any timepoint investigated. Despite the lack of active caspase-3, the newly formed stratum granulosum and the regressing periderm contained cells positive in the terminal deoxynucleotidyl transferase-mediated fluorescein-dUTP nick end labeling assay, indicating that nuclear DNA was degraded without activation of caspase-3, thereby arguing against a proteolytic function of caspase-3 in embryonic KC differentiation. By contrast, caspase-14 increased in abundance from embryonic day 14.5 (E14.5) onwards and consistently localized to the suprabasal layers of fetal epidermis. The caspase-14 pro-enzyme was processed into its catalytic subunits, a step required for enzyme activity, on day E17.5, coinciding with SC formation. Thus, processing of procaspase-14 is not confined to air-exposed mature skin but also occurs during epidermal development in utero. In summary, this study demonstrates that caspase-14, but not caspase-3 activation coincides temporally and spatially with embryonic KC differentiation, suggesting a role for caspase-14 in terminally differentiated KC.     

Synchronous luminescence: a simple technique for the analysis of hydrolysis activity of the fragile histidine triad protein

Human fragile histidine triad (FHIT) protein has dinucleoside 5,5-P₁,P_n-polyphosphates hydrolysis activity, with AMP being one of the reaction products. Application of synchronous luminescence (SL) spectroscopy, in which both excitation and emission wavelengths are scanned simultaneously while a constant wavelength interval is maintained between them, was investigated for detection of the enzymatic activity of the FHIT protein. Ability of SL to identify reaction components, AMP production and its increase as a result of increase in substrate concentration and inhibition of the hydrolysis activity by ZnCl₂ are demonstrated.     

Toll/IMD signal pathways mediate cellular immune responses via induction of intracellular PLA 2 expression

Phospholipase A₂ (PLA₂) hydrolyzes fatty acids from phospholipids at the sn‐2 position. Two intracellular PLA₂s, iPLA₂A and iPLA₂B, have been found in Spodoptera exigua. Both are calcium‐independent cellular PLA₂. Their orthologs have been found in other insects. These two iPLA₂s are different in ankyrin motif of N terminal region. The objective of this study was to determine whether Toll/immune deficiency (IMD) signal pathways could mediate cellular immune responses via induction of iPLA₂ expression. Both iPLA ₂s were expressed in all developmental stages of S. exigua, showing the highest expression in the adult stage. During larval stage, hemocyte is the main tissue showing expression of these iPLA₂s. Both iPLA₂s exhibited similar expression patterns after immune challenge with different microbial pathogens such as virus, bacteria, and fungi. Promoter component analysis of orthologs encoded in S. frugiperda indicated nuclear factor‐κB‐ and Relish‐responsible elements on their promoters, suggesting their expression in S. exigua under Toll/IMD immune signaling pathways. RNA interference (RNAi) of MyD88 or Pelle under Toll pathway suppressed inducible expression levels of both iPLA₂s in response to Gram‐positive bacteria containing Lys‐type peptidoglycan or fungal infection. In contrast, RNAi against Relish under IMD pathway suppressed both iPLA₂s in response to infection with Gram‐negative bacteria. Under RNAi conditions, hemocytes significantly lost cellular immune response measured by nodule formation. However, addition of arachidonic acid (a catalytic product of PLA₂) rescued such immunosuppression. These results suggest that Toll/IMD signal pathways can mediate cellular immune responses via eicosanoid signaling by inducing iPLA₂ expression.     

A Novel Syntaxin 6‐Interacting Protein,SHIP164, Regulates Syntaxin 6‐Dependent Sorting from Early Endosomes

Membrane fusion is dependent on the function of SNAREs and their α‐helical SNARE motifs that form SNARE complexes. The Habc domains at the N‐termini of some SNAREs can interact with their associated SNARE motif, Sec1/Munc18 (SM) proteins, tethering proteins or adaptor proteins, suggesting that they play an important regulatory function. We screened for proteins that interact with the Habc domain of Syntaxin 6, and isolated an uncharacterized 164‐kDa protein that we named SHIP164. SHIP164 is part of a large (∼700 kDa) complex, and interacts with components of the Golgi‐associated retrograde protein (GARP) tethering complex. Depletion of GARP subunits or overexpression of Syntaxin 6 results in a redistribution of soluble SHIP164 to endosomal structures. Co‐overexpression of Syntaxin 6 and SHIP164 produced excessive tubulation of endosomes, and perturbed the transport of cation‐independent mannose‐6‐phosphate receptor (CI‐MPR) and transferrin receptor. Thus, we propose that SHIP164 functions in trafficking through the early/recycling endosomal system.     

The Association between Mycobacterium Tuberculosis Genotype and Drug Resistance in Peru

BackgroundThe comparison of Mycobacterium tuberculosis bacterial genotypes with phenotypic, demographic, geospatial and clinical data improves our understanding of how strain lineage influences the development of drug-resistance and the spread of tuberculosis.MethodsTo investigate the association of Mycobacterium tuberculosis bacterial genotype with drug-resistance. Drug susceptibility testing together with genotyping using both 15-loci MIRU-typing and spoligotyping, was performed on 2,139 culture positive isolates, each from a different patient in Lima, Peru. Demographic, geospatial and socio-economic data were collected using questionnaires, global positioning equipment and the latest national census.ResultsThe Latin American Mediterranean (LAM) clade (OR 2.4, p<0.001) was significantly associated with drug-resistance and alone accounted for more than half of all drug resistance in the region. Previously treated patients, prisoners and genetically clustered cases were also significantly associated with drug-resistance (OR''s 2.5, 2.4 and 1.8, p<0.001, p<0.05, p<0.001 respectively).ConclusionsTuberculosis disease caused by the LAM clade was more likely to be drug resistant independent of important clinical, genetic and socio-economic confounding factors. Explanations for this include; the preferential co-evolution of LAM strains in a Latin American population, a LAM strain bacterial genetic background that favors drug-resistance or the "founder effect" from pre-existing LAM strains disproportionately exposed to drugs.     

Changes in serum parameters associated with iron metabolism in male rat exposed to lead

Due to the severe hazardous influences of lead (Pb²⁺) on iron-related diseases, the effects of Pb²⁺ on serum parameters associated with iron metabolism have been studied in this project. Male Wistar rats weighing 200–250 g were treated with Pb²⁺ for the short and long period of times. The animals received daily intraperitoneal injection of 100 mg Pb²⁺ kg^?1 body weight (BW) for 5 days and 4 mg?kg^?1 BW of Pb²⁺ for 30 and 45 days, respectively. The results show that when animals were treated with both low and high concentrations of Pb²⁺, serum iron concentration decreased markedly, by 23.2, 32.8, and 39.9 %, while the sera TIBC and transferrin concentrations increased significantly (p?<?0.05). Following short- and long-term exposures to Pb²⁺, the percentage of serum transferrin saturation was also decreased in comparison with the untreated control group (p?<?0.05). Concentrations of serum copper and ceruloplasmin following Pb²⁺ treatments also reduced significantly (p?<?0.05). The percentage of hematocrit and hemoglobin levels was reduced (p?<?0.05) in all Pb²⁺-treated animals in comparison with the controls. These results suggest that Pb²⁺ changes the serum parameters related to iron metabolism, which may play an important role in producing iron-related diseases.