Immobilization of tyrosinase within polyacrylamide gels

The enzyme tyrosinase (E.G. 1.14.18.1) has been immobilized in a polyacrylamide gel and intermittently assayed for enzyme activity over a period of 19 days using phenol as the substrate. The results of these studies indicate that the immobilized enzyme could be incorporated into a system to detect phenol and related compounds that are found in industrial effluents and as surface water contaminants.     

Aldolases of the lactic acid bacteria

Reciprocal qualitative and quantitative immunological experiments employing an anti-Pediococcus cerevisiae aldolase serum confirmed many of the interspecific relationships demonstrated previously among lactic acid bacteria with antisera prepared against the Streptococcus faecalis fructose diphosphate aldolase. The extent of immunological relatedness observed between the Lactobacillus and Pediococcus aldolases was markedly greater than that noted between Pediococcus and Streptococcus aldolases indicating that the pediococci share closer phylogenetic ties with the rod-shaped lactobacilli than with their spherical counterparts in the streptococci. In addition to confirming the existence of definitive, but distant, relationships between the lactic acid bacteria and certain gram positive nonsporeforming anaerobes, immunological cross-reactivity was also demonstrated between the pediococcal aldolases and those of Aerococcus viridans.This paper is dedicated with deepest appreciation to Prof. Roger Y. Stanier on the occasion of his 60th birthday in token of what his friendship and guidance have meant to me. — J. L.     

Cycloheximide resistance in Chinese hamster cells. II. Induction of Chm resistance in Chinese hamster cells by N-nitrosomethylurea.

Mutant Chinese hamster ovarian (CHO) cells with a resistance to 7-10(-7) and 8-10(-7) M cycloheximide (CHM) were induced at mutation rates of 1.9-5.2-10(-3) and 1.6-1.8-10(-3) respectively after treatment with N-nitrosomethylurea (NMU) at 100 mug/ml. The induced mutation rates differed by two orders of magnitude from the spontaneous rate of mutation to CHM resistance.     

A radioimmunoassay of plasma estradiol-17beta with the use of polyethylene glycol to separate free and antibody-bound hormone

A radioimmunoassay for plasma estradiol-17β was developed using polyethylene glycol to separate free from antibody-bound hormone. Specificity for estradiol-17β was achieved by a modified celite microcolunm procedure in which estradiol was.separated from interfering estrogens, including estrone. Using trace ³H-estradiol to monitor procedural losses, the method was shown to be sensitive and accurate. Intra- and inter-assay coefficient of variation of the method was 8.7 and 10.6%, respectively. Polyethylene glycol used for antibody precipitation appears to be a generally applicable method for steroid hormone radioimmunoassays. The simplicity, precision and rapid analysis, coupled with its lack of time dependence and ease in automation, makes this a convenient and practical method.     

Fusing VE-Cadherin to α-Catenin Impairs Fetal Liver Hematopoiesis and Lymph but Not Blood Vessel Formation

We have recently shown that genetic replacement of VE-cadherin by a VE-cadherin–α-catenin fusion construct strongly impairs opening of endothelial cell contacts during leukocyte extravasation and induction of vascular permeability in adult mice. Here we show that this mutation leads to lethality at midgestation on a clean C57BL/6 background. Investigating the reasons for embryonic lethality, we observed a lack of fetal liver hematopoiesis and severe lymphedema but no detectable defects in blood vessel formation and remodeling. As for the hematopoiesis defect, VE-cadherin–α-catenin affected neither the generation of hematopoietic stem and progenitor cells (HSPCs) from hemogenic endothelium nor their differentiation into multiple hematopoietic lineages. Instead, HSPCs accumulated in the fetal circulation, suggesting that their entry into the fetal liver was blocked. Edema formation was caused by disturbed lymphatic vessel development. Lymphatic progenitor cells of VE-cadherin–α-catenin-expressing embryos were able to leave the cardinal vein and migrate to the site of the first lymphatic vessel formation, yet subsequently, these cells failed to form large lumenized lymphatic vessels. Thus, stabilizing endothelial cell contacts by a covalent link between VE-cadherin and α-catenin affects recruitment of hematopoietic progenitors into the fetal liver and the development of lymph but not blood vessels.     

A diverse uncultivated microbial community is responsible for organic matter degradation in the Black Sea sulphidic zone

Organic matter degradation in marine environments is essential for the recycling of nutrients, especially under conditions of anoxia where organic matter tends to accumulate. However, little is known about the diversity of the microbial communities responsible for the mineralization of organic matter in the absence of oxygen, as well as the factors controlling their activities. Here, we determined the active heterotrophic prokaryotic community in the sulphidic water column of the Black Sea, an ideal model system, where a tight coupling between carbon, nitrogen and sulphur cycles is expected. Active microorganisms degrading both dissolved organic matter (DOM) and protein extracts were determined using quantitative DNA stable isotope probing incubation experiments. These results were compared with the metabolic potential of metagenome-assembled genomes obtained from the water column. Organic matter incubations showed that groups like Cloacimonetes and Marinimicrobia are generalists degrading DOM. Based on metagenomic profiles the degradation proceeds in a potential interaction with members of the Deltaproteobacteria and Chloroflexi Dehalococcoidia. On the other hand, microbes with small genomes like the bacterial phyla Parcubacteria, Omnitrophica and of the archaeal phylum Woesearchaeota, were the most active, especially in protein-amended incubations, revealing the potential advantage of streamlined microorganisms in highly reduced conditions.     

Population structure and adaptation of a bacterial pathogen in California grapevines

Xylella fastidiosa subsp. fastidiosa causes Pierce's disease of grapevine (PD) and has been present in California for over a century. A singly introduced genotype spread across the state causing large outbreaks and damaging the grapevine industry. This study presents 122 X. fastidiosa subsp. fastidiosa genomes from symptomatic grapevines, and explores pathogen genetic diversity associated with PD in California. A total of 5218 single-nucleotide polymorphisms (SNPs) were found in the dataset. Strong population genetic structure was found; isolates split into five genetic clusters divided into two lineages. The core/soft-core genome constituted 41.2% of the total genome, emphasizing the high genetic variability of X. fastidiosa genomes. An ecological niche model was performed to estimate the environmental niche of the pathogen within California and to identify key climatic factors involved in dispersal. A landscape genomic approach was undertaken aiming to link local adaptation to climatic factors. A total of 18 non-synonymous polymorphisms found to be under selective pressures were correlated with at least one environmental variable highlighting the role of temperature, precipitation and elevation on X. fastidiosa adaptation to grapevines in California. Finally, the contribution to virulence of three of the genes under positive selective pressure and of one recombinant gene was studied by reverse genetics.     

Peptide Bond Cleavage by Ni(II) Ions within the Nuclear Localization Signal Sequence

Nickel is harmful to humans, being both carcinogenic and allergenic. However, the mechanisms of this toxicity are still unresolved. We propose that Ni(II) ions disintegrate proteins by hydrolysis of peptide bonds preceding the Ser/Thr‐Xaa‐His sequences. Such sequences occur in nuclear localization signals (NLSs) of human phospholipid scramblase 1, Sam68‐like mammalian protein 2, and CLK3 kinase. We performed spectroscopic experiments showing that model nonapeptides derived from these NLSs bind Ni(II) at physiological pH. We also proved that these sequences are prone to Ni(II) hydrolysis. Thus, the aforementioned NLSs may be targets for nickel toxicity. This implies that Ni(II) ions disrupt the transport of some proteins from cytoplasm to cell nucleus.