Gracilaria conferta and its epiphytes: 3. Allelopathic inhibition of the red seaweed byUlva cf.lactuca

In a previous study (Svirski et al., 1993), it was found that growth inhibition ofGracilaria spp., when cultured in the presence ofUlva cf.lactuca, was not due to shading or nutrient depletion, but seemed to be caused by competition for inorganic carbon or some type of allelopathy. In the present study, we attempted to differentiate between these two possible influences by (1) growing the two algae in biculture under various conditions, but keeping inorganic carbon levels constant and measuring net photosynthesis, respiration and growth rates, and by (2) measuring growth rates ofGracilaria spp. in the presence of extracts derived from media previously used to growUlva cf.lactuca.Both net photosynthesis and growth rates ofGracilaria spp. in biculture were inhibited, despite CO₂ (and also HCO₃ ^–) levels being kept constantly high in the culture media. It is likely that these responses were due to markedly enhanced rates of dark respiration inGracilaria spp. when grown together withUlva cf.lactuca. Growth ofGracilaria spp. was also inhibited by extracts derived from seawater in whichUlva cf.lactuca had previously been grown. The strong inhibition by ethyl acetate and chloroform extracts indicate an allelopathic effect onGracilaria spp.     

Simple procedure for purification of diphtheria toxin and separation of its fragments by hydrophobic chromatography

Diphtheria toxin and fragment B bind to hydrocarbon-coated agaroses. Fragment A of the toxin is not adsorbed to such resins. Using Seph-C₄, the toxin and fragment B can be eluted from the column after adsorption by increasing the ionic strength of the eluent. The toxin is also eluted from the Seph-C₆ column, but fragment B is eluted only in the denatured form. Purification of the toxin can be achieved simply by passing the growth medium supernatant through a small size Seph-C₆ column and eluting the toxin by 0.1 m NaCl. The fragments of diphtheria toxin obtained after mild trypsin treatment can be separated purely on a Seph-C₄ column. The hydrophobic chromatography system may thus serve as a tool for purification of the toxin and its fragments: it may also be useful in large-scale preparations.     

Chloramphenicol binding site of an fi− R-factor-specified variant of chloramphenicol acetyltransferase

Chloramphenicol acetyltransferase (EC 2.3.1.28) specified by the fi^? R-factor (type II) is highly sensitive to sulfhydryl reagents. When this variant was treated with stoichiometric amounts of 2, 2′dithiobispyridine, 90% of the enzymatic activity was lost with concomitant introduction of 0.9to 1.0 thiopyridine groups per mole of enzyme protomer. In the presence of stoichiometric amounts of the substrate, chloramphenicol, the enzyme was neither inactivated nor modified by the sulfhydryl reagents. Acetyl-coenzyme A exerted no protective effects when present in the reaction mixture. The enzyme was also inactivated by cyanylation with a stoichiometric amount of 2-nitro-5-thiocyanobenzoic acid. Labeling native type II enzyme with iodo[¹⁴C]acetamide and subsequently subjecting it to peptic digestion yielded one radioactive peptide. This cysteine-containing peptide had the same sequence as that found near the cysteine close to the chloramphenicol binding site of the commonly occurring type 1 enzyme. In conclusion, this cysteine residue is essential for the catalytic activity of both types of enzyme and is located in or near the chloramphenicol binding site. It also seems that the cysteine in type II is more sensitive to sulfhydryl reagents than the homologous cysteine in type I, probably because it is more available for modification.     

Collection of Continuous Rotation MicroED Data from Ion Beam-Milled Crystals of Any Size

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BMI may overestimate the prevalence of obesity among women of lower socioeconomic status

Objective: Our objective was to examine gender differences in height and weight associated with socioeconomic status (SES) and the consequent effect on body mass index in a multiethnic society. Research Methods and Procedures: A cross‐sectional study, the First Israeli National Health and Nutrition Survey, was performed on a representative population sample of 3246 adults 25 to 64 years of age, between the years 1999 to 2001. Height and weight were measured, and BMI and other weight‐height indices were calculated. SES was assessed by income and education. Results: Age‐adjusted height was significantly lower at lower levels of SES among both women and men (p < 0.001). As opposed to men, women of lower SES were heavier than those of higher SES, and the mean age‐adjusted weight was 4.6 kg higher among those of lower SES (p < 0.001). Thus, using the standard index of BMI, the prevalence of obesity was significantly higher among shorter women. Discussion: In this group of Israeli adults, the unfavorable effect of low SES on BMI was evident among women, partly due to their decreased height combined with increased weight common in this socioeconomic sector. Since BMI is only partly independent of height, it may overestimate the prevalence of obesity among women of lower SES. Alternative measures for classifying obesity in the lower SES groups that put less emphasis on height may be considered and studied.     

Regulation of the Drosophila ubiquitin ligase DIAP1 is mediated via several distinct ubiquitin system pathways

Inhibitors of apoptosis proteins (IAPs) suppress cell death by inactivating proapoptotic regulators, and therefore play important roles in controlling apoptosis in normal and malignant cells. Many IAPs are ubiquitin ligases, and their activity is mediated via ubiquitination and subsequent degradation of their targets. Here we corroborate a previous observation that DIAP1 (Drosophila IAP1) can be degraded via a two-step mechanism: (i) limited caspase-mediated cleavage and (ii) degradation of the released fragment via the ubiquitin N-end rule pathway. Yet, we demonstrate that this pathway is not the only one involved in DIAP1 degradation, and the intact protein can be degraded independent of prior caspase cleavage. Importantly, this mode of degradation does not require the RING-finger-mediated autoubiquitinating activity of DIAP1, believed to target many RING-finger E3s for self-destruction. Our preliminary data suggest that DIAP2 mediates DIAP1 degradation, suggesting a novel regulatory loop within the apoptotic pathway. Studying the role of the autoubiquitinating activity of DIAP1, we demonstrate that it does not involve formation of Lys48-based polyubiquitin chains, but probably chains linked via Lys63. Our preliminary data suggest that the autoubiquitination serves to attenuate the ligase activity of DIAP1 towards its exogenous substrates.     

Tension applied through the Dam1 complex promotes microtubule elongation providing a direct mechanism for length control in mitosis

In dividing cells, kinetochores couple chromosomes to the tips of growing and shortening microtubule fibres and tension at the kinetochore-microtubule interface promotes fibre elongation. Tension-dependent microtubule fibre elongation is thought to be essential for coordinating chromosome alignment and separation, but the mechanism underlying this effect is unknown. Using optical tweezers, we applied tension to a model of the kinetochore-microtubule interface composed of the yeast Dam1 complex bound to individual dynamic microtubule tips. Higher tension decreased the likelihood that growing tips would begin to shorten, slowed shortening, and increased the likelihood that shortening tips would resume growth. These effects are similar to the effects of tension on kinetochore-attached microtubule fibres in many cell types, suggesting that we have reconstituted a direct mechanism for microtubule-length control in mitosis.     

A genetic screen identifies genes essential for development of myelinated axons in zebrafish

The myelin sheath insulates axons in the vertebrate nervous system, allowing rapid propagation of action potentials via saltatory conduction. Specialized glial cells, termed Schwann cells in the PNS and oligodendrocytes in the CNS, wrap axons to form myelin, a compacted, multilayered sheath comprising specific proteins and lipids. Disruption of myelinated axons causes human diseases, including multiple sclerosis and Charcot-Marie-Tooth peripheral neuropathies. Despite the progress in identifying human disease genes and other mutations disrupting glial development and myelination, many important unanswered questions remain about the mechanisms that coordinate the development of myelinated axons. To address these questions, we began a genetic dissection of myelination in zebrafish. Here we report a genetic screen that identified 13 mutations, which define 10 genes, disrupting the development of myelinated axons. We present the initial characterization of seven of these mutations, defining six different genes, along with additional characterization of mutations that we have described previously. The different mutations affect the PNS, the CNS, or both, and phenotypic analyses indicate that the genes affect a wide range of steps in glial development, from fate specification through terminal differentiation. The analysis of these mutations will advance our understanding of myelination, and the mutants will serve as models of human diseases of myelin.