A Stoichiometry Driven Universal Spatial Organization of Backbones of Folded Proteins: Are there Chargaff's Rules for Protein Folding?

Abstract

Protein folding is at least a six decade old problem, since the times of Pauling and Anfinsen. However, rules of protein folding remain elusive till date. In this work, rigorous analyses of several thousand crystal structures of folded proteins reveal a surprisingly simple unifying principle of backbone organization in protein folding. We find that protein folding is a direct consequence of a narrow band of stoichiometric occurrences of amino-acids in primary sequences, regardless of the size and the fold of a protein. We observe that “preferential interactions” between amino-acids do not drive protein folding, contrary to all prevalent views. We dedicate our discovery to the seminal contribution of Chargaff which was one of the major keys to elucidation of the stoichiometry-driven spatially organized double helical structure of DNA.     

In vivo Liver Endocytosis Followed by Purification of Liver Cells by Liver Perfusion

The liver is the metabolic center of the mammalian body and serves as a filter for the blood. The basic architecture of the liver is illustrated in figure 1 in which more than 85% of the liver mass is composed of hepatocytes and the remaining 15% of the cellular mass is composed of Kupffer cells (KCs), stellate cells (HSCs), and sinusoidal endothelial cells (SECs). SECs form the blood vessel walls within the liver and contain specialized morphology called fenestrae within in the cytoplasm. Fenestration of the cytoplasm is the appearance of holes (˜100 μm) within the cells so that the SECs act as a sieve in which most chylomicrons, chylomicron remnants and macromolecules, but not cells, pass through to the hepatocytes and HSCs ¹ (Fig. 1). Due to the lack of a basement membrane, the gap between the SECs and hepatocytes form the Space of Disse. HSCs occupy this space and play a prominent role in regulation and response to injury, storage of retinoic acid and immunoregulation of the liver ².SECs are among the most endocytically active cells of the body displaying an array of scavenger receptors on their cell surface ³. These include SR-A, Stabilin-1 and Stabilin-2. Generally, small colloidal particles less than 230 nm and macromolecules in buffer phase are taken up by SECs, whereas, large particles and cellular debris is endocytosed (phagocytosed) by KCs ⁴. Thus, the bulk clearance of extracellular material such as the glycosaminoglycans from blood is largely dependent on the health and endocytic functions of SECs ^5,6. For example, an increase in blood hyaluronan levels is indicative of liver disease ranging from mild to more severe forms ⁷.With the exception of one report ⁸, there are no immortalized SEC cell lines in existence. Even this immortalized cell line is de-differentiated in that it does not express scavenger receptors that are present on primary SECs (our data, not shown). All cell biological studies must be performed on primary cells obtained freshly from the animal. Unfortunately, SECs dedifferentiate under standard culture conditions and must be used within 1 or 2 days upon isolation from the animal. Differentiation of SECs is marked by the expression of Stabilin-2 or HARE receptor ⁹ , CD31, and the presence of cytoplasmic fenestration ¹. Differentiation of SECs can be extended by the addition of VEGF in culture media or by culturing cells in hepatocyte conditioned medium ^10,11.In this report, we will demonstrate the endocytic activity of SECs in the intact organ using radio-labeled heparin for hyaluronan for the SEC-specific Stabilin-2 receptor. We will then purify hepatocytes and SECs from the perfused liver to measure endocytosis.     

Influence of incubation period on the plasmid content of proliferating Nocardioides sp.

S. SANDHYA. 1996. Nocardioides sp., isolated for organic sulphur degradation, harbours catabolic plasmid pSB1 of molecular weight 34.2 kb. A correlation was noticed between the plasmid content and degradation of the organic sulphur compound dibenzothiophene (DBT). The maximum oxidation of DBT was only after 64 h, during which the content of plasmid DNA was also optimum.