Methanopyrus kandleri topoisomerase V contains three distinct AP lyase active sites in addition to the topoisomerase active site

Topoisomerase V (Topo-V) is the only topoisomerase with both topoisomerase and DNA repair activities. The topoisomerase activity is conferred by a small alpha-helical domain, whereas the AP lyase activity is found in a region formed by 12 tandem helix-hairpin-helix ((HhH)₂) domains. Although it was known that Topo-V has multiple repair sites, only one had been mapped. Here, we show that Topo-V has three AP lyase sites. The atomic structure and Small Angle X-ray Scattering studies of a 97 kDa fragment spanning the topoisomerase and 10 (HhH)₂ domains reveal that the (HhH)₂ domains extend away from the topoisomerase domain. A combination of biochemical and structural observations allow the mapping of the second repair site to the junction of the 9th and 10th (HhH)₂ domains. The second site is structurally similar to the first one and to the sites found in other AP lyases. The 3rd AP lyase site is located in the 12th (HhH)₂ domain. The results show that Topo-V is an unusual protein: it is the only known protein with more than one (HhH)₂ domain, the only known topoisomerase with dual activities and is also unique by having three AP lyase repair sites in the same polypeptide.     

Pathogenic LMNA variants disrupt cardiac lamina-chromatin interactions and de-repress alternative fate genes

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Preclinical pharmacokinetic characterization of an adipose tissue-targeting monoclonal antibody in obese and non-obese animals

Target receptor levels can influence pharmacokinetics (PK) or pharmacodynamics (PD) of monoclonal antibodies (mAbs), and can affect drug development of this class of molecules. We generated an effector-less humanized bispecific antibody that selectively activates fibroblast growth factor receptor (FGFR)1 and βKlotho receptor, a FGF21 receptor complex highly expressed in both white and brown adipocytes. The molecule shows cross-species binding with comparable equilibrium binding affinity (Kd) for human, cynomolgus monkey, and mouse FGFR1/βKlotho. To understand the PK/PD relationship in non-obese and obese animals, we evaluated the adipose tissue distribution of the antibody, serum exposures, and an associated PD marker (high-molecular-weight adiponectin), in both non-obese and obese mice and monkeys. Antibody uptake into fat tissue was found to be higher on a per gram basis in non-obese animals compared to obese animals. Since obesity has been reported to be associated with reduced expression of FGFR1 and βKlotho receptor in white adipose tissues in mice, our results suggest that the distribution in adipose tissues was influenced by target expression levels. Even so, the overall dose-normalized serum exposures were comparable between non-obese and obese mice and monkeys, suggesting that adipose tissue uptake plays a limited role in overall systemic PK determination. It remains to be determined if and how obesity and receptor expression in humans influence the PK and PD profile of this novel therapeutic candidate.     

Distribution of metal ions in the subcellular fractions of several rat brain areas.

The levels of Cu, Fe, Zn, Mg and Ca in the synaptosomal myelin and mitochondrial fractions of the rat brain were determined quantitatively by means of the atomic absorption spectroscopic method. In the whole brain the concentrations of the metals were present in the three different fractions in the following range; Cu, 0.46-1.76; Fe, 0.24-3.51; Zn, 0.12-0.93; Mg, 1.08-1.41 and Ca, 1.28-3.56 μg/mg tissue protein. Analyses of the subcellular organelles prepared from different areas of the brain indicated that the concentrations of Cu and Zn were relatively larger in the hypothalamus and lower in cerebellum in comparison with other areas. On treatment of the rats with the metal chelating agents i.e., EDA, EDDHA and HBED intracisternally, it was found that the distribution of the subcellular metals was markedly affected.     

Physico-chemical characterisation and molecular organisation of the collagen from the skin of an air-breathing fish (Ophiocephalus striatus)

Collagen has been prepared from the skin of an air-breathing Indian fish(Ophiocephalus striatus) by extraction with cold 0.5M acetic acid and purification by alternate precipitation with NaCl and dialysis against 0.02M Na₂HPO₄. The purified collagen was characterised with respect to physico-chemical properties, amino acid composition and chromatography of the denatured collagen. The fish collagen has a higher shrinkage temperature and denaturation temperature compared to that of the allied teleosts living in exclusively aquatic medium. These differences could possibly be reflections of the response to the rigours of the environment. As found for other vertebrate collagens, the fish collagen contains two kinds of single chains the α₁ and α₂ chains as revealed by sodium dodecyl sulphate-polyacrylamidegel electrophoresis and carboxymethylcellulose chromatography.     

The possible relevance of autoxidative glycosylation in glucose mediated alterations of proteins: An in vitro study on myofibrillar proteins

The present work was carried out to examine the role of glycation and transition metal catalysed autoxidation of sugars in glucose-mediated alterations of myofibrillar proteins. Myofibrils were prepared from rat skeletal muscle and incubated with 1) sugar alone 2) sugar and micromolar concentrations of transition metals (Cu²⁺ or Fe³⁺) 3) transition metals alone and the control remained without sugar or transition metals. A significant increase in extent of glycation and decrease in ATPase activity of myofibrils incubated under autoxidative conditions were observed over the other three incubations. Reducing agent 2-mercaptoethanol was highly effective in preventing the alterations induced by glucoxidation, compared to EDTA and aminoguanidine, suggesting the involvement of thiol group oxidation in the reduced function of the protein. Free radical scavengers like catalase, benzoic acid and mannitol were also effective in preventing glucose mediated alterations. Although a high concentration of glucose alone has an insignificant effect on myofibrils in vitro, the results from the present work suggest that glucose in combination with transition metals could lead to functional alterations of myofibrils, and this process by generating free radicals may contribute to the overall complications of diabetes and aging.