The Modular Organisation and Stability of a Thermostable Family 10 Xylanase

The thermophilic marine bacterium Rhodothermus marinus produces a modular family 10 xylanase (Xyn10A). It consists of two N-terminal family 4 carbohydrate binding modules (CBMs) followed by a domain of unknown function (D3), and a catalytic module (CM) flanked by a small fifth domain (D5) at its C-terminus. Several truncated mutants of the enzyme have been produced and characterised with respect to biochemical properties and stability. Multiple calcium binding sites are shown to be present in the two N-terminal CBMs and recent evidence suggests that the third domain of the enzyme also has the ability to bind the same metal ligand. The specific binding of Ca²⁺ was demonstrated to have a pronounced effect on thermostability as shown by differential scanning calorimetry and thermal inactivation studies. Furthermore, deletion mutants of the enzyme were less stable than the full-length enzyme suggesting that module interactions contributed to the stability of the enzyme. Finally, recent evidence indicates that the fifth domain of Xyn10A is a novel type of module mediating cell-attachment.     

Engineering of Hydrolysis Reaction Specificity in the Transglycosylase Cyclodextrin Glycosyltransferase

Abstract

Cyclodextrin glycosyltransferase (CGTase) is a member of the α-amylase family, a large group of enzymes that act on α-glycosidic bonds in starch and related compounds. Over twenty different reaction and product specificities have been found in this family. Although three-dimensional structure elucidation and the biochemical characterization of site-directed mutants have yielded a detailed insight into the mechanism of bond cleavage, the variation in reaction and product specificity is far from understood. This article gives an overview of recent developments in the undersanding and engineering of transglycosylation and hydrolysis specificity in CGTase, which is one of the best-studied α-amylase family enzymes.     

Conformational insights on the molecular recognition processes of carbohydrate molecules by proteins and enzymes: A 3D view by using NMR

This review focuses, in a non-exhaustive manner, on the essential structural and conformational features of protein–carbohydrate interactions and on some applications of NMR spectroscopy to deal with this topic from different levels of complexity.     

Chronic stress and high sucrose intake cause distinctive morphometric effects in the adrenal glands of post-weaned rats

We investigated the effects of chronic stress combined with high sucrose intake on the morphology of the adrenal glands in young rats. Male Wistar rats were fed a standard chow diet and allocated into control (C; tap water), chronic restraint stress (St), 30% sucrose diet (S30) and 30% sucrose diet + chronic restraint stress (S30 + St) groups. St consisted of 1 h daily sessions, 5 days/week for 4 weeks. Chronic stress reduced the thickness of the zona glomerulosa (ZG) and zona fasciculata (ZF) in both right and left glands; the thickness of the zona reticularis (ZR) was increased in the right gland. Cell density was greater in the ZF and medulla of both right and left glands, whereas cell density increased in the ZR of only the left gland. The percentage of small cells was lower in the ZG, whereas more large cells were found in the left gland. A similar result was obtained for the ZF, ZR and medulla in both right and left glands. Chronic stress increased the area covered by blood vessels in the ZR of the right gland, but decreased the area in the ZR of the left gland. The area covered by blood vessels was reduced in the medulla of both right and left glands in rats subjected to chronic stress. Infiltration of immune cells was increased by chronic stress in all layers of the cortex of the left gland, but was reduced in the medulla of the right gland. A high sucrose diet reduced the thickness of the medulla in the left gland. Cell proliferation increased in the ZG of the right gland and the weight of the right adrenal gland increased. Reduced cell proliferation in the ZG of the left gland was associated with a reduction in the area covered by blood vessels. In addition, the area covered by blood vessels decreased in the medulla of both glands. Our findings demonstrate that exposure to chronic stress during early life causes morphometric changes in adrenal glands.     

2-Methylwyosine,a Nucleoside With Restricted Anti Conformation in the East Region Enforced by Nucleobase Moiety Modification: Synthesis and Conformational Analysis by NMR and Molecular Dynamics

GRAPHICAL ABSTRACT

We synthesized a new 2-methyl derivative of wyosine using a multistep procedure starting from guanosine. We examined different synthetic paths and optimized the conditions for each step. Based on MD calculations and analysis of the ³ J _HH and J _C1′H1′ of the ribose moiety, we discovered that the sugar part adopted conformation specific for the East region rarely occurring in solution. This unusual conformational preference is probably due to steric repulsions between the methyl group at position 2 and the 5′-CH₂OH group. We observed that N-glycosidic bond stability weakened 14-fold upon the introduction of the methyl group in position 2 compared with wyosine.     

Structure and Conformation of 1-β-D-Ribo Furanosyl Pyridin-2-one-5-Carboxamide: An Anti-Inflammatory Agent

The pyrimidine nucleoside, 1-β-D-ribofuranosyl pyridine-2-one-5-carboxamide, is an anti inflammatory agent used in the treatment of adjuvant-induced arthritis. It is the 2-one isomer of 1-β-D-ribofuranosyl pyridine-4-one 5-carboxamide, an unusual nucleoside isolated from the urine of patients with chronic myelogenic leukemia and an important cancer marker. Crystals of 1-β-D-ribofuranosyl pyridine-2-one-5-carboxamide are monoclinic, space group C2, with the cell dimensions a = 31.7920(13), b = 4.6872 (3), c = 16.1838(11), β = 93.071(3)°, V = 2408.2(2) ų, D_calc = 1.496 mg/m³ and Z = 8 (two molecules in the asymmetric unit). The structure was obtained by the application of direct methods to diffractometric data and refined to a final R value of 0.050 for 1669 reflections with I ≥ 3σ. The nucleoside exhibits an anti conformation across the glycosidic bond (χ_CN = ?15.5°, ?18.9°), a C3 ′- endo C2 ′ -exo [³ ₂T] ribose pucker and g⁺ across the C(4 ′)-C(5 ′) exocyclic bond. The amino group of the carboxamide group is distal from the 2-one and lacks the intramolecular hydrogen bonding found in the related 2-one molecule. Nuclear magnetic resonance studies shows also an anti conformation across the glycosidic bond but the solution conformation of the furanose ring is not the same as that found in the solid state.     

Clerocidin-mediated DNA footprinting discriminates among different G-quadruplex conformations and detects tetraplex folding in a duplex environment

Background

G-quadruplexes are polymorphic non-canonical nucleic acid conformations involved both in physiological and pathological processes. Given the high degree of folding heterogeneity and comparable conformational stabilities, different G-quadruplex forms can occur simultaneously, hence rendering the use of basic instrumental methods for structure determination, like X-ray diffraction or NMR, hardly useful. Footprinting techniques represent valuable and relatively rapid alternative to characterize DNA folding. The natural diterpenoid clerocidin is an alkylating agent that specifically reacts at single-stranded DNA regions, with different mechanisms depending on the exposed nucleotide.

Methods

Clerocidin was used to footprint G-quadruplex structures formed by telomeric and oncogene promoter sequences (c-myc, bcl-2, c-kit2), and by the thrombin binding aptamer.

Results

The easy modulability of CL reactivity towards DNA bases permitted to discriminate fully and partially protected sites, highlights stretched portions of the G-quadruplex conformation, and discriminate among topologies adopted by one sequence in different environmental conditions. Importantly, CL displayed the unique property to allow detection of G-quadruplex folding within a duplex context.

Conclusions

CL is a finely performing new tool to unveil G-quadruplex arrangements in DNA sequences under genomically relevant conditions.

General significance

Nucleic acid G-quadruplex structures are an emerging research field because of the recent indication of their involvement in a series of key biological functions, in particular in regulation of proliferation-associated gene expression. The use of clerocidin as footprinting agent to identify G-quadruplex structures under genomically relevant conditions may allow detection of new G-quadruplex-based regulatory regions.     

Changes in the biochemical composition of stored potato tubers during pathogenesis by Rhizoctonia bataticola

The nutrient contents of the two cultivare of Potato‐Irish Cobbler and Red Pontiac were quantitatively altered by Rhizoctonia bataticola during pathogenesis. The glucose content of both cultivare increased whereas sucrose, maltose and fructose contents decreased appreciably in quantity during the period of incubation. The total protein and lipid contents of both cultivare were also lowered quantitatively by the rot‐causing organism. The depletion of total carbohydrate, protein and lipid contents, was more in Irish Cobbler cultivar than in Red Pontiac cultivar.     

Genetic polymorphisms and protein structures in growth hormone,growth hormone receptor,ghrelin, insulin-like growth factor 1 and leptin in Mehraban sheep

The somatotropic axis, the control system for growth hormone (GH) secretion and its endogenous factors involved in the regulation of metabolism and energy partitioning, has promising potentials for producing economically valuable traits in farm animals. Here we investigated single nucleotide polymorphisms (SNPs) of the genes of factors involved in the somatotropic axis for growth hormone (GH1), growth hormone receptor (GHR), ghrelin (GHRL), insulin-like growth factor 1 (IGF-I) and leptin (LEP), using polymerase chain reaction–single-strand conformation polymorphism (PCR–SSCP) and DNA sequencing methods in 452 individual Mehraban sheep. A nonradioactive method to allow SSCP detection was used for genomic DNA and PCR amplification of six fragments: exons 4 and 5 of GH1; exon 10 of GH receptor (GHR); exon 1 of ghrelin (GHRL); exon 1 of insulin-like growth factor-I (IGF-I), and exon 3 of leptin (LEP). Polymorphisms were detected in five of the six PCR products. Two electrophoretic patterns were detected for GH1 exon 4. Five conformational patterns were detected for GH1 exon 5 and LEP exon 3, and three for IGF-I exon 1. Only GHR and GHRL were monomorphic. Changes in protein structures due to variable SNPs were also analyzed. The results suggest that Mehraban sheep, a major breed that is important for the animal industry in Middle East countries, has high genetic variability, opening interesting prospects for future selection programs and preservation strategies.