Structure of Haze Forming Proteins in White Wines: Vitis vinifera Thaumatin-Like Proteins

Grape thaumatin-like proteins (TLPs) play roles in plant-pathogen interactions and can cause protein haze in white wine unless removed prior to bottling. Different isoforms of TLPs have different hazing potential and aggregation behavior. Here we present the elucidation of the molecular structures of three grape TLPs that display different hazing potential. The three TLPs have very similar structures despite belonging to two different classes (F2/4JRU is a thaumatin-like protein while I/4L5H and H2/4MBT are VVTL1), and having different unfolding temperatures (56 vs. 62°C), with protein F2/4JRU being heat unstable and forming haze, while I/4L5H does not. These differences in properties are attributable to the conformation of a single loop and the amino acid composition of its flanking regions.     

Met326Ile aminoacid polymorphism in the human p85 alpha gene has no major impact on early insulin signaling in type 2 diabetes.

Class I alpha phosphatidylinositol (PI) 3-kinase is an important enzyme in the early insulin signaling cascade, and plays a key role in insulin-mediated glucose transport. Despite extensive investigation, the genes responsible for the development of the common forms of type 2 diabetes remain unknown. This study was performed to identify variants in the coding region of p85 alpha, the regulatory subunit of PI 3-kinase. Fibroblasts from skin biopsies from type 2 diabetics and controls were established to address this issue. P85 alpha cDNA was sequenced, and a single point mutation at codon 326 was found. This mutation resulted in a homozygous missense amino acid change Met --> Ile in one subject with type 2 diabetes and heterozygous variant in two other diabetic patients and one with severe insulin resistance. Interestingly, those patients revealed an impaired insulin-mediated insulin receptor substrate (IRS)-1 binding to p85 alpha without any alteration in IRS-2/p85 alpha association. Furthermore, IRS-1, IRS-2, p85 alpha and MAPK protein contents were not significantly changed, and neither were MAPK or Akt phosphorylation. We conclude from our data that this variant may have only minor impact on signaling events; however, in combination with variants in other genes encoding signaling proteins, this may have a functional impact on early insulin signaling.     

No evidence for linkage of autosomal dominant proximal spinal muscular atrophies to chromosome 5q markers

Summary Two recent articles have reported the linkage of a gene for recessive spinal muscular atrophy (SMA) on the chromosome region 5q11.2–13.3. Our data show no linkage of the dominantly inherited forms of SMA to this chromosome region.     

Molecular characterization and genetic mapping of two clusters of genes encoding chlorophyll a/b-binding proteins in Lycopersicon esculentum (tomato)

We have constructed a tomato genomic library in the λ Charon 4 phage vector. The library was screened with a pea cDNA probe encoding a chlorophyll a/b-binding protein (CAB), and several recombinant phages containing tomato CAB genes were isolated and characterized by restriction mapping, heteroduplex analysis and nucleotide sequencing. Two phages with overlapping segments of the tomato genome contain a total of four CAB genes, all arranged in tandem. A third phage contains three CAB genes, two arranged in tandem and one in opposite orientation, and an additional, truncated CAB gene. Genetic mapping experiments showed that the four CAB genes on the first two phages belong to a locus, previously designated Cab-1, on chromosome 2. The CAB genes from the third phage belong to the Cab-3 locus on chromosome 3. Complete sequence determination of two CAB genes, one from each locus, and additional sequence determination of about 50% of each of the other five CAB genes showed that each gene within a CAB locus is more similar to other CAB genes in the same locus than it is to the CAB genes from the second locus. Furthermore, the polypeptides encoded by Cab-1 genes diverge significantly from those encoded by Cab-3 genes in the domains of transit peptide and the N terminus of the mature polypeptide but are essentially identical in the rest of the sequence.     

Chitinase activity in the digestive tract of the cod, Gadus morhua (L.)

The study was made to determine if enzymatic degradation of chitin occurs in the digestive tract of the cod, Gadus morhua . The method employed corresponds to the end product measurement of Jeuniaux (1966), using 'native' chitin as the substrate. The following results were obtained.

• (1) 

  Chitinolytic enzyme of high activity is present in enzyme solutions from the stomach contents, gastric mucosa and intestinal contents.
• (2) 

  Lower chitinase activities are found in samples of the intestinal mucosa and the pyloric caeca.
• (3) 

  The optimum pH ranges for the action of the enzymes in the stomach and the intestine differ: 4.5–5.1 and 5.1–6.5, respectively.
• (4) 

  The role of chitin-decomposing bacteria is discussed, based on bacterial numbers and pH conditions in the digestive tract. The existence of two different enzyme systems is indicated.

     

Die Primärwurzel von Zea mays L.

Zusammenfassung An Gewächshauskulturen kann gezeigt werden, daß die Primärwurzel von Zea mays L. während der ganzen Lebensperiode der Pflanze perenniert. Da auch bei Phoenix dactylifera L. eine solche Tendenz zu erkennen ist, ist es wahrscheinlich, daß weitere Monocotylen diese Art der Bewurzelung zeigen. Es ließe sich daraus ein Maiswurzeltyp der heterogenen Radication bei den Monocotylen ableiten, bei dem primäres und sproßbürtiges Wurzelsystem dauernd nebeneinander bestehen.

---

The primary root of Zea mays L.

Summary Until now it has been presumed that the primary root of Zea mays L. dies very soon after the formation of the seedling. On the basis of this presumption maize root-system development has been used as an example for monocotyledons (z.B. Troll, 1937; Rauh, 1950).After passing the seedling stage, monocotyledons are said to have shoot-born roots only. There are a few hints in the literature of exceptions to this principle known in the case of some palms (Falkenberg, 1876; Fitting, 1954; Kausch, unpublished). In this paper it is shown that the primary root of maize does not die off soon, but remains living during the entire vegetation period. Investigations were made within the greenhouse, where in the stage of flowering of the plants the primary root reached downwards into the soil as far as 1.60 m (see also Fig. 2). There are also some observations in the open field showing that here too the primary root remains living until the plant dies in autumn.Nevertheless Zea mays has sekundäre Homorhizie and heterogene Radication (Troll, 1949). However, there is surely a large group within the monocotyledons which is capable of keeping the primary root system along with shoot-born roots. Of this group it may be said that it is of the Mais-Wurzel-Typ.

     

Effects of Sirolimus treatment on patients with β-Thalassemia: Lymphocyte immunophenotype and biological activity of memory CD4+ and CD8+ T cells

Inhibitors of the mammalian target of rapamycin (mTOR) have been proposed to improve vaccine responses, especially in the elderly. Accordingly, testing mTOR inhibitors (such as Sirolimus) and other geroprotective drugs might be considered a key strategy to improve overall health resilience of aged populations. In this respect, Sirolimus (also known as rapamycin) is of great interest, in consideration of the fact that it is extensively used in routine therapy and in clinical studies for the treatment of several diseases. Recently, Sirolimus has been considered in laboratory and clinical studies aimed to find novel protocols for the therapy of hemoglobinopathies (e.g. β-Thalassemia). The objective of the present study was to analyse the activity of CD4⁺ and CD8⁺ T cells in β-Thalassemia patients treated with Sirolimus, taking advantages from the availability of cellular samples of the NCT03877809 clinical trial. The approach was to verify IFN-γ releases following stimulation of peripheral blood mononuclear cells (PBMCs) to stimulatory CEF and CEFTA peptide pools, stimulatory for CD4⁺ and CD8⁺ T cells, respectively. The main results of the present study are that treatment of β-Thalassemia patients with Sirolimus has a positive impact on the biological activity and number of memory CD4⁺ and CD8⁺ T cells releasing IFN-γ following stimulation with antigenic stimuli present in immunological memory. These data are to our knowledge novel and in our opinion of interest, in consideration of the fact that β-Thalassemia patients are considered prone to immune deficiency.     

RosettaDDGPrediction for high-throughput mutational scans: From stability to binding

Reliable prediction of free energy changes upon amino acid substitutions (ΔΔGs) is crucial to investigate their impact on protein stability and protein–protein interaction. Advances in experimental mutational scans allow high-throughput studies thanks to multiplex techniques. On the other hand, genomics initiatives provide a large amount of data on disease-related variants that can benefit from analyses with structure-based methods. Therefore, the computational field should keep the same pace and provide new tools for fast and accurate high-throughput ΔΔG calculations. In this context, the Rosetta modeling suite implements effective approaches to predict folding/unfolding ΔΔGs in a protein monomer upon amino acid substitutions and calculate the changes in binding free energy in protein complexes. However, their application can be challenging to users without extensive experience with Rosetta. Furthermore, Rosetta protocols for ΔΔG prediction are designed considering one variant at a time, making the setup of high-throughput screenings cumbersome. For these reasons, we devised RosettaDDGPrediction, a customizable Python wrapper designed to run free energy calculations on a set of amino acid substitutions using Rosetta protocols with little intervention from the user. Moreover, RosettaDDGPrediction assists with checking completed runs and aggregates raw data for multiple variants, as well as generates publication-ready graphics. We showed the potential of the tool in four case studies, including variants of uncertain significance in childhood cancer, proteins with known experimental unfolding ΔΔGs values, interactions between target proteins and disordered motifs, and phosphomimetics. RosettaDDGPrediction is available, free of charge and under GNU General Public License v3.0, at https://github.com/ELELAB/RosettaDDGPrediction .