The signature amidase from Sulfolobus solfataricus belongs to the CX3C subgroup of enzymes cleaving both amides and nitriles. Ser195 and Cys145 are predicted to be the active site nucleophiles

The signature amidase from the extremophile archeum Sulfolobus solfataricus is an enantioselective enzyme that cleaves S-amides. We report here that this enzyme also converts nitriles in the corresponding organic acid, similarly to the well characterized amidase from Rhodococcus rhodochrous J1. The archaeal and rhodococcal enzymes belong to the signature amidases and contain the typical serine-glycine rich motif. They work at different optimal temperature, share a high sequence similarity and both contain an additional CX3C motif. To explain their dual specificity, we built a 3D model of the structure of the S. solfataricus enzyme, which suggests that, in addition to the classical catalytic Ser-cisSer-Lys, a putative additional Cys-cisSer-Lys catalytic site, likely to be responsible for nitrile hydrolysis, is present in these proteins. The results of random and site-directed mutagenesis experiments, as well as inhibition studies support our hypothesis.     

Glycoprotein reglucosylation

Proteins following the secretory pathway acquire their proper tertiary and in certain cases also quaternary structures in the endoplasmic reticulum (ER). Incompletely folded species are retained in the ER and eventually degraded. One of the molecular mechanisms by which cells achieve this conformational sorting is based on monoglucosylated N-glycans (Glc1Man5-9GlcNAc2) present on nascent glycoproteins in the ER. This chapter discusses two of the steps that regulate the abundance of such N-glycan structures, including glycoprotein deglucosylation (by glucosidase II) and reglucosylation (by the UDP-Glc:glycoprotein glucosyltransferase), as well as an overview of methods to evaluate the N-glycans prevalent during glycoprotein biogenesis in the ER.     

Asparaginyl deamidation in two glutamate dehydrogenase isoenzymes from Saccharomyces cerevisiae

The non-enzymatic deamidation of asparaginyl residues is a major source of spontaneous damage of several proteins under physiological conditions. In many cases, deamidation and isoaspartyl formation alters the biological activity or stability of the native polypeptide. Rates of deamidation of particular residues depend on many factors including protein structure and solvent exposure. Here, we investigated the spontaneous deamidation of the two NADP-glutamate dehydrogenase isoenzymes from Saccharomyces cerevisiae, which have different kinetic properties and are differentially expressed in this yeast. Our results show that Asn54, present in Gdh3p but missing in the GDH1-encoded homologue, is readily deamidated in vitro under alkaline conditions. Relative to the native enzyme, deamidated Gdh3p shows reduced protein stability. The different deamidation rates of the two isoenzymes could explain to some extent, the relative in vivo instability of the allosteric Gdh3p enzyme, compared to that of Gdh1p. It is thus possible that spontaneous asparaginyl modification could play a role in the metabolic regulation of ammonium assimilation and glutamate biosynthesis.     

Phenanthrenoids from the wetland Juncus acutus

Nine 9,10-dihydrophenanthrenes, three phenanthrenes and a related pyrene have been isolated from the wetland plant Juncus acutus. The structures have been attributed by means of their spectral data and chemical correlation. 5-(1-Ethoxy-ethyl)-2-hydroxy-7-methoxy-1,8-dimethyl-9,10-dihydrophenanthrene and 5-(1-phytoxy-ethyl)-2-hydroxy-7-methoxy-1,8-dimethyl-9,10-dihydrophenanthrene, 2,7-dihydroxy-1-methyl-5-vinylphenanthrene, 2,7-dimethoxy-1,6-dimethyl-5-vinylphenanthrene and 2,7-dihydroxy-1,6-dimethylpyrene are described for the first time. Many of the compounds showed in vitro phytotoxicity against Selenastrum capricornutum, a microalga used in aquatic tests.     

Towards a morphogenetic perspective on cancer.

The purpose of this paper is to present a critique of the current view that reduces cancer to a cellular problem caused by specific gene mutations and to propose, instead, that such a problem might become more intelligible, if it is understood as a phenomenon which results from the breakdown of the morphological plan or Gestalt of the organism. Such an organism, in Aristotelian terms, is characterised for presenting a specific morpho or logos (form) and for having a telos (end) to fulfill. A malignant tumour represents an entity separated from both, the organic logos and the organic telos. According to the basic postulates of Semiophysics--a blend of Aristotelian physics and Catastrophe Theory developed by René Thom--an organism is a source (original) form individuated by a dominant pregnancy which corresponds to its morphogenetic field. Here it is suggested that cancer in aged individuals may result from the progressive exhaustion of the developmental constraints that regulate the process of ontogeny, that is expected to go from the fertilised non-differentiated zygote to the mature fully-developed organism, because there is no further point ahead in the developmental pathway past the reproductive age. Cancer in young individuals (before their reproductive maturity) may then be consequence of premature derangement of such fundamental developmental constraints. In all cases the result is the loss of morphological coherence within the organism. Thus representing a conflict between an organised morphology (the organism) and a part of such a morphology that drifts towards an amorphous state (the tumour).     

MUCILAGINOUS CAPSULE ADSORPTION AND INTRACELLULAR UPTAKE OF COPPER BY KIRCHNERIELLA APERTA (CHLOROCOCCALES)1

The purpose of the present investigation was to evaluate possible ecological and physiological functions of mucilaginous capsules produced by the freshwater algae Kirchneriella aperta Teiling (Chlorococcales) as related to copper ions. All experiments were performed using synthetic media under laboratory‐controlled conditions. Copper interactions were investigated by distinguishing between adsorption onto the mucilaginous material present at the surface of the cells, intracellular uptake, and differentiation between total dissolved copper and free copper ions in the culture medium. Kirchneriella aperta is sensitive to copper, as revealed by a 96‐h EC₅₀ value of 10 ^{? 9.22} M Cu^{2 +} . We demonstrated that the mucilaginous capsules were able to sequester copper ions from the medium through a passive mechanism, thus providing the cell with a mechanism able to postpone the toxic effects of copper. The organic material that diffuses into the test medium as well as the mucilaginous capsules produced by K. aperta both effectively complex copper; thus, toxicity must be related to free copper ions and not the total dissolved copper concentration in the medium.     

LC-MS/MS quantification of short-chain acyl-CoA's in Escherichia coli demonstrates versatile propionyl-CoA synthetase substrate specificity

Aims: This paper utilized quantitative LC‐MS/MS to profile the short‐chain acyl‐CoA levels of several strains of Escherichia coli engineered for heterologous polyketide production. To further compare and potentially expand the levels of available acyl‐CoA molecules, a propionyl‐CoA synthetase gene from Ralstonia solanacearum (prpE‐RS) was synthesized and expressed in the engineered strain BAP1. Methods and Results: Upon feeding propionate, the engineered E. coli strains had increased the levels of both propionyl‐ and methylmalonyl‐CoA of 6‐ to 30‐fold and 3·7‐ to 6·8‐fold, respectively. Expression of prpE‐RS resulted in no significant increases in acetyl‐, butyryl‐ and propionyl‐CoA when fed the corresponding substrates (sodium acetate, butyrate or propionate). More interesting, however, were the results from strain BAP1 engineered for native prpE overexpression, which indicated increases in the same range of acyl‐CoA formation. Conclusions: The increased acyl‐CoA levels across the strains profiled in this study reflect the genetic modifications implemented for improved polyketide production and also indicate flexibility of the native PrpE. Significance and Impact of the Study: The results provide direct evidence of enhanced acyl‐CoA levels correlating to those strains engineered for polyketide biosynthesis. This information and the inherent flexibility of the native PrpE enzyme support future efforts to characterize, engineer and extend acyl‐CoA precursor supply for additional heterologous biosynthetic attempts.     

Deploying MMEJ using MENdel in precision gene editing applications for gene therapy and functional genomics

Gene-editing experiments commonly elicit the error-prone non-homologous end joining for DNA double-strand break (DSB) repair. Microhomology-mediated end joining (MMEJ) can generate more predictable outcomes for functional genomic and somatic therapeutic applications. We compared three DSB repair prediction algorithms – MENTHU, inDelphi, and Lindel – in identifying MMEJ-repaired, homogeneous genotypes (PreMAs) in an independent dataset of 5,885 distinct Cas9-mediated mouse embryonic stem cell DSB repair events. MENTHU correctly identified 46% of all PreMAs available, a ∼2- and ∼60-fold sensitivity increase compared to inDelphi and Lindel, respectively. In contrast, only Lindel correctly predicted predominant single-base insertions. We report the new algorithm MENdel, a combination of MENTHU and Lindel, that achieves the most predictive coverage of homogeneous out-of-frame mutations in this large dataset. We then estimated the frequency of Cas9-targetable homogeneous frameshift-inducing DSBs in vertebrate coding regions for gene discovery using MENdel. 47 out of 54 genes (87%) contained at least one early frameshift-inducing DSB and 49 out of 54 (91%) did so when also considering Cas12a-mediated deletions. We suggest that the use of MENdel helps researchers use MMEJ at scale for reverse genetics screenings and with sufficient intra-gene density rates to be viable for nearly all loss-of-function based gene editing therapeutic applications.