Molecular evidence demonstrating the basidiomycetous fungus Cryptococcus curvatus is the dominant microbial eukaryote in sediment at the Kuroshima Knoll methane seep

The Kuroshima Knoll, located in the southern Ryukyu Arc, is known to actively bubble with gas containing methane and hydrogen sulfide from numerous fissures in the large carbonate pavement. Although ecological studies regarding macrobenthos and bacteria from Kuroshima Knoll have been intensively conducted, the community structure and ecological importance of microbial eukaryotes (protists) have not yet been investigated. In the present study, we directly extracted DNA from sediment of the Kuroshima Knoll at a depth of 640 m and constructed genetic libraries of PCR-amplified eukaryotic small-subunit ribosomal DNA (SSU rDNA). Although the SSU rDNA sequences of several types of benthic foraminifers were retrieved from the surface of the sediment, all other sequences (just below the sediment surface to approximately 9 cm below sediment surface) were derived from the basidiomycetous yeast Cryptococcus curvatus. Furthermore, sequences of the internal transcribed spacer of rDNA (ITS-rDNA) retrieved from the same sediment were identical to that of C. curvatus originating from terrestrial habitats. The diversity of microbial eukaryotes in the Kuroshima Knoll sediment seems to be extremely low and significantly different from that of other marine environments previously reported.     

Severe thrombocytosis and anemia associated with celiac disease in a young female patient: a case report

Introduction

Platelet counts exceeding 1.000 × 10³/μl are usually considered secondary to another cause, particularly to chronic myeloproliferative disease (CMPD). Reactive thrombocytosis due to iron deficiency rarely exceeds platelet counts of 700 × 10³/μl.

Case presentation

Here we report the case of a young woman presenting with clinical signs of severe anemia. Laboratory findings confirmed an iron-deficiency anemia associated with severe thrombocytosis of 1703 × 10³/μl. Macroscopic gastrointestinal and genitourinary tract bleeding was excluded. The excessive elevation of platelets, slightly elevated lactate dehydrogenase and slightly elevated leukocytes along with the absence of other inflammation parameters raised the suspicion of an underlying hematological disease. However, bone marrow evaluation could not prove the suspected diagnosis of a CMPD, especially essential thrombocythemia (ET). In the further clinical course the platelet count returned to normal after raising the hemoglobin to a level close to normal range with erythrocyte transfusion, and normalization of serum iron and decline of erythropoietin. Finally, following small bowel biopsy, despite the absence of typical clinical signs, celiac disease was diagnosed. After discharge from hospital the patient was commenced on a gluten-free diet and her hemoglobin almost completely normalized in the further follow-up period.

Conclusion

This case illustrates the rare constellation of an extreme thrombocytosis most likely secondary to iron deficiency due to celiac disease. This represents, to the best of the authors' knowledge, the highest reported platelet count coincident with iron deficiency. A potential mechanism for the association of iron-deficiency anemia and thrombocytosis is discussed. Even in the presence of 'atypically' high platelets one should consider the possibility of reactive thrombocytosis. Extreme thrombocytosis could emerge in the case of iron deficiency secondary to celiac disease.

     

Yeast silencers create domains of nuclease-resistant chromatin in an SIR4-dependent manner

Previous analysis of the repression of the silent mating type loci in Saccharomyces cerevisiae has linked the mechanism of silencing to the formation of a chromatin domain at the silenced loci. In this study, a TRP1 reporter gene was used to examine changes in chromatin structure in a neutral environment. This enabled the chromatin structure organized by yeast silencers to be compared directly with changes effected by the yeast α2 repressor. It was found that silencers mediate the formation of lengthy nuclease-resistant domains on the DNA, rather than specifically positioning nucleosomes over promoter regions as the α2 repressor does. Silencing at the TRP1 reporter gene closely resembled silencing at the HMR and HML loci. Repression of the test gene was optimal when two silencers flanking the reporter gene were used, mimicking the situation at the silent loci. In addition, both repression of the reporter gene and the formation of nuclease-resistant chromatin domains was SIR4 dependent. Received: 31 October 1996; in revised form: 6 March 1997 / Accepted: 25 March 1997     

Ultrastructure and synthesis of the extracellular matrix of Pisaster ochraceus embryos preserved by freeze substitution

When asteroid embryos cryoprotected with propylene glycol are rapidly frozen in liquid propane and freeze substituted with ethanol, preservation of the cells and extracellular matrix (ECM) is excellent. The basal lamina, although thicker and less well defined than in conventionally fixed embryos, demonstrates a region of decreased density just below the cells that corresponds to the lamina lucida and a lamina densa. The former region is often occupied by fibrous material. In addition, as was previously described in conventionally fixed tissues, the basal lamina of the ectoderm is generally thicker and more substantial than that of the endoderm, reinforcing an earlier suggestion that the structure of the basal lamina is different in different regions of the embryo. The ECM of the blastocoel consists of thin “twig-like” elements that form a loose meshwork evenly distributed throughout the blastocoel. Bundles of 20 nm fibers, located within the meshwork, are oriented parallel to the base of the cells of the stomodeum. In the long axis of the embryo, similar fibers are present in the dorsal aspect of the animal between the stomach and the ectoderm and radiate out from the esophagus crossing the region between it and the ectoderm. Immunocytochemical work with three different monoclonal antibodies shows that glycoprotein molecules, synthesized in the Golgi apparatus, are also secreted here and form part of the matrix structure. The results suggest that the blastocoel is filled with a gel-like material reinforced with bundles of 20-nm fibers. The manner in which the observed arrangement could contribute to the development and maintainence of the shape of the embryo is discussed. J Morphol 232:133–153, 1997. © 1997 Wiley-Liss, Inc.     

Binding of chimeric metal-binding green fluorescent protein to lipid monolayer

Membrane-based bioanalytical devices for metal determination using green fluorescent protein as the sensor molecule may be a useful future biomimetic material. However, in order to develop such a device, it is necessary first to understand the interaction of the protein with lipid membranes. Thus we have investigated the interaction between chimeric cadmium-binding green fluorescent proteins (CdBPGFPs) and lipid monolayers, using a film-balance technique complemented with epifluorescence microscopy. The binding avidity was monitored from the surface pressure vs. area isotherms or from the measured increase in the lateral pressure upon injection of the chimeric CdBPGFPs beneath the lipid monolayer. Increased fluidization as well as expansion of the surface area were shown to depend on the concentration of the CdBPGFPs. The kinetics of the protein-induced increase in lateral pressure was found to be biphasic. The chimeric CdBPGFPs possessed high affinity to the 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) monolayer with a dissociation constant of K_d=10^–8M. Epifluorescence measurements showed that this affinity is due to the presence of the Cd-binding peptide, which caused the GFP to incorporate preferentially to the liquid phase and defect part of the rigid domain at low interfacial pressure. At high compression, the Cd-binding peptide could neither incorporate nor remain in the lipid core. However, specific orientation of the chimeric CdBPGFPs underneath the air–water interface was achieved, even under high surface pressure, when the proteins were applied to the metal-chelating lipid-containing surfaces. This specific binding could be controlled reversibly by the addition of metal ions or metal chelator. The reversible binding of the chimeric CdBPGFPs to metal-chelating lipids provided a potential approach for immobilization, orientation and lateral organization of a protein at the membrane interface. Furthermore, the feasibility of applying the chelator lipids for the codetermination of metal ions with specific ligands was also revealed. Our finding clearly demonstrates that a strong interaction, particularly with fluid lipid domains, could potentially be used for sensor development in the future.Abbreviations GFP green fluorescent protein - CdBPGFPs cadmium-binding green fluorescent protein - DPPC 1,2-dipalmitoyl-sn-glycero-3-phosphocholine - AAS atomic absorption spectrometry - Cd²⁺ cadmium (II) - Zn²⁺ zinc (II) - Cu²⁺ copper (II) - Ni²⁺ nickel (II) - E. coli Escherichia coli - NTA-DOGS 1,2-dioleoyl-sn-glycero-3-(N-(5-amino-1-carboxypentyl iminodiacetic acid) succinyl) - His6GFP hexahistidine green fluorescent protein - CdBP4GFP four-repeat cadmium-binding peptide green fluorescent protein - His6CdBP4GFP hexahistidine four-repeat cadmium-binding peptide green fluorescent protein - IMAC immobilized-metal-affinity chromatography - PBS phosphate-buffered saline - mN/m millinewton per metre - le liquid expanded - lc liquid condensed - PE phosphatidyl ethanolamine - PI phosphatidyl inositol - NTA nitrilotriacetic acid - EDTA ethylenediamine tetraacetic acid - RESA ring-infected erythrocyte surface antigen - CdBP cadmium-binding peptide     

Degradation of biomolecules in bones: effects of the biological forensics as an example of the stability of isotope ratios in collagen

Modern bone samples were experimentally degraded by incubation into water at increased temperature and examined in terms of their collagen content, the stable C and N isotopic ratios, and the molar C/N ratio. The same analyses were carried out with archaeological human bone of varying age (300 up to 8000 years). The experimentally degraded samples exhibited changes of the collagen's integrity, which influence the stable isotope ratios. In the case of the archaeological material, a correlation between stable delta13C- and delta15N-values and collagen content could be demonstrated. The molar C:N ratio was no suitable criterion for the assessment of the state of preservation of extractable collagen.     

Seed and gland morphology in Euphorbia (Euphorbiaceae) with focus on their systematic and phylogenetic importance, a case study in Iranian highlands

A comprehensive study based on gland and seed micromorphology in Euphorbia (Euphorbiaceae) for species distributed in Iranian highlands is presented. A total of 86 species were studied. The gland structure was examined by direct field observations. Taxonomically important characters of glands were observed and measured: size, texture, shape, color, and horns. For species out of Iran herbarium materials were studied. Seed characteristics were examined using scanning electron microscopy (SEM) as well as dissecting light microscopy. Significant features are: seed size, seed shape, presence of caruncle, shape of caruncle, and seed surface ornamentation. A phylogenetic study using Maximum Parsimony (MP) and Bayesian Inference (BI) was performed based on sequences of nuclear DNA internal transcribed spacers (ITS) for selected species representing the main clades known in Euphorbia and with special focus on the species distributed in Iranian highlands. ITS sequences for 20 accessions representing 19 species are provided for the first time, and 48 accessions of 47 species were used from GenBank. The topologies of both analyses were congruent. The results indicate: (1) four main clades with high supports in subgen. Esula which are appropriate to be recognized at sectional rank. (2) E. larica is nested within clade A including few members of subgen. Rhizanthium and is closely related to sect. Balsamis, which is suggested here to be transferred from subgen. Esula into subgen. Rhizanthium. (3) E. osyridea of the monotypic subsect. Osyrideae is closely related to E. buhsei and to the members of sect. Esula. Tracing morphological characters on the phylogenetic tree shows that several morphological characters, such as seed ornamentation applied in previous subgeneric classification of the subgen. Esula, are homoplasious, but the gland structure and capsule surface characters are more reliable for classification purposes.     

Neuromodulatory unpaired median neurons in the New Zealand tree weta, Hemideina femorata

Wetas are ancient Gondwanan orthopterans (Anostostomatidae) with many species endemic to New Zealand. Like all Orthoptera they possess efferent neuromodulatory dorsal unpaired median (DUM) neurons, with bilaterally symmetrical axons, that are important components of motor networks. These neurons produce overshooting action potentials and are easily stimulated by a variety of external mechanosensory stimuli delivered to the body and appendages. In particular, stimulation of the antennae, mouth parts, tarsi and femora of the legs, abdomen, cerci and ovipositor is very effective in activating DUM neurons in the metathoracic ganglion of wetas. In addition, looming visual stimuli or light on-, light off-stimuli excite many metathoracic DUM neurons. These DUM sensory reflex pathways remain viable after the prothoracic to subesophageal connective is cut, whereas in locusts such reflex pathways are interrupted by the ablation. This suggests that, in wetas, sensory reflex pathways for DUM activation are organized in a less centralized fashion than in locusts, and may therefore reflect a plesiomorphic evolutionary state in the weta. In addition, many weta DUM neurons exhibit slow rhythmic bursting which also persists following the connective ablation.     

Inactivation of the elongation factor Tu by mosquitocidal toxin-catalyzed mono-ADP-ribosylation

The mosquitocidal toxin (MTX) produced by Bacillus sphaericus strain SSII-1 is an approximately 97-kDa single-chain toxin which contains a 27-kDa enzyme domain harboring ADP-ribosyltransferase activity and a 70-kDa putative binding domain. Due to cytotoxicity toward bacterial cells, the 27-kDa enzyme fragment cannot be produced in Escherichia coli expression systems. However, a nontoxic 32-kDa N-terminal truncation of MTX can be expressed in E. coli and subsequently cleaved to an active 27-kDa enzyme fragment. In vitro the 27-kDa enzyme fragment of MTX ADP-ribosylated numerous proteins in E. coli lysates, with dominant labeling of an approximately 45-kDa protein. Matrix-assisted laser desorption ionization-time-of-flight mass spectrometry combined with peptide mapping identified this protein as the E. coli elongation factor Tu (EF-Tu). ADP ribosylation of purified EF-Tu prevented the formation of the stable ternary EF-Tuaminoacyl-tRNAGTP complex, whereas the binding of GTP to EF-Tu was not altered. The inactivation of EF-Tu by MTX-mediated ADP-ribosylation and the resulting inhibition of bacterial protein synthesis are likely to play important roles in the cytotoxicity of the 27-kDa enzyme fragment of MTX toward E. coli.