Mammary Gland Reprogramming: Metalloproteinases Couple Form with Function

The adult mammary structure provides for the rapid growth, development, and immunological protection of the live-born young of mammals through its production of milk. The dynamic remodeling of the branched epithelial structure of the mammary gland in response to physiological stimuli that allow its programmed branching morphogenesis at puberty, cyclical turnover during the reproductive cycle, differentiation into a secretory organ at parturition, postlactational involution, and ultimately, regression with age is critical for these processes. Extracellular metalloproteinases are essential for the remodeling programs that operate in the tissue microenvironment at the interface of the epithelium and the stroma, coupling form with function. Deregulated proteolytic activity drives the transition of a physiological mammary microenvironment into a tumor microenvironment, facilitating malignant transformation.Milk production is an evolutionary survival strategy that allows the rapid growth and development of live-born young, as well as being a defining trait of mammals. Milk production at parturition and not at other times conserves valuable energy resources of the mother. It also provides immunological protection to the offspring. Thus, the response of the adult mammary structure to changes in systemic hormones as well as locally derived factors is to expand the ductal network into a milk-producing gland rapidly and to tear it down again when its function is no longer required. The origin of the mammary gland during evolution including its link with the immune system has inspired considerable interest but remains speculative (Oftedal 2002; Vorbach et al. 2006; Widelitz et al. 2007; McClellan et al. 2008). Although the branched epithelial structure of the mammary gland varies in composition and complexity among mammals, the alveolar acinus, its cellular secretory unit, and the tubular ducts, which channel milk for delivery through the teat, have been conserved in mammals.The concept of form and function first arose from the study of mammary epithelial cells and has become a cornerstone in biology, because it applies universally to other parenchymal units (Lee et al. 1984). Here, epithelial cells retain apical-basal polarity and shape by establishing physical contacts with the structural matrix and neighboring cells, and build an adequate form to enable their function of milk production in response to appropriate stimuli (Boudreau and Bissell 1998). The mammary gland encounters constant physiological demands during the female lifespan. To maintain its function, it must repeatedly reacquire its fundamental form with the preservation of cell types, ratios, differentiation state, and matrix integrity. This requires remodeling programs that initially allow development of the mammary gland at puberty, cyclical turnover during the reproductive cycle, differentiation into a secretory organ at parturition, postlactational involution, and ultimately, regression with age. In this article, we describe the essential features of remodeling programs that generally operate at the parenchymal-stromal interface in the mammary tissue microenvironment, and highlight the critical role of extracellular proteolysis in coupling form with function. We also discuss how deregulated protease activity facilitates the transition of a physiological mammary microenvironment into a tumor microenvironment.Our understanding of the mammary gland has been enriched by the use of the mouse as an experimental system, and, thus, observations from murine genetic models, loss- and gain-of-function studies, as well as transplantation assays form the basis of this article. Although the mouse has become an integral part of investigations, there are notable differences between human and murine mammary biology (Cardiff and Wellings 1999). The mammary epithelial ductal system in humans differs considerably with respect to its branching pattern, the stromal, adipocyte, and extracellular matrix (ECM) content, as well as the hormonal triggers that provide the mammotrophic stimuli. Therefore species-specific differences must be considered while making generalizations for the mammary gland.     

Cardiogel: A Nano-Matrix Scaffold with Potential Application in Cardiac Regeneration Using Mesenchymal Stem Cells

3-Dimensional conditions for the culture of Bone Marrow-derived Stromal/Stem Cells (BMSCs) can be generated with scaffolds of biological origin. Cardiogel, a cardiac fibroblast-derived Extracellular Matrix (ECM) has been previously shown to promote cardiomyogenic differentiation of BMSCs and provide protection against oxidative stress. To determine the matrix composition and identify significant proteins in cardiogel, we investigated the differences in the composition of this nanomatrix and a BMSC-derived ECM scaffold, termed as ‘mesogel’. An optimized protocol was developed that resulted in efficient decellularization while providing the maximum yield of ECM. The proteins were sequentially solubilized using acetic acid, Sodium Dodecyl Sulfate (SDS) and Dithiothreitol (DTT). These proteins were then analyzed using surfactant-assisted in-solution digestion followed by nano-liquid chromatography and tandem mass spectrometry (nLC-MS/MS). The results of these analyses revealed significant differences in their respective compositions and 17 significant ECM/matricellular proteins were differentially identified between cardiogel and mesogel. We observed that cardiogel also promoted cell proliferation, adhesion and migration while enhancing cardiomyogenic differentiation and angiogenesis. In conclusion, we developed a reproducible method for efficient extraction and solubilization of in vitro cultured cell-derived extracellular matrix. We report several important proteins differentially identified between cardiogel and mesogel, which can explain the biological properties of cardiogel. We also demonstrated the cardiomyogenic differentiation and angiogenic potential of cardiogel even in the absence of any external growth factors. The transplantation of Bone Marrow derived Stromal/Stem Cells (BMSCs) cultured on such a nanomatrix has potential applications in regenerative therapy for Myocardial Infarction (MI).     

Prediction of cystine connectivity using SVM

One of the major contributors to protein structures is the formation of disulphide bonds between selected pairs of cysteines at oxidized state. Prediction of such disulphide bridges from sequence is challenging given that the possible combination of cysteine pairs as the number of cysteines increases in a protein. Here, we describe a SVM (support vector machine) model for the prediction of cystine connectivity in a protein sequence with and without a priori knowledge on their bonding state. We make use of a new encoding scheme based on physico-chemical properties and statistical features (probability of occurrence of each amino acid residue in different secondary structure states along with PSI-blast profiles). We evaluate our method in SPX (an extended dataset of SP39 (swiss-prot 39) and SP41 (swiss-prot 41) with known disulphide information from PDB) dataset and compare our results with the recursive neural network model described for the same dataset.     

Drug, dosage, activity, studies of antimalarials by physical methods--II

Studies pertaining to drug-DNA interactions in treating a disease efficiently have taken an important place in recent times. Murthy and colleagues were active in correlating the drug activity, with physical parameters like refractivity, susceptibility, molecular electron ionization cross-section and the dosage. The molecular polarizability, diamagnetic susceptibility and molecular electron ionization cross section Q have been evaluated. An analysis of Q in the light of the data available on plasma protein binding, bio availability, Log P and half-Life show semblance of regular dependence of Q on them and hence an effort is made to bring this dependence into a regular mathematical relationship. The dosage of each drug is calculated. A critical look at the results arrived on Q and dosages reveal that a highly active drug with large Q need to be monitored in very small quantities and any minute increase in dosage is resulting in unwanted toxic effects and vice versa. The algebraic formulae enable one to calculate the dosages theoretically from the value of Q and other parameters and the calculated dosage through the formulae agreed favorably well with suggested dosages. For example, in primaquine phosphate, the calculated dosage is 30 mg per day against the suggested practical dosage of 26.3 mg per day. A similar observation is made in mepacrine with theoretical dosage of 60 mg per day as against the suggested practical dosage of 100 mg per day. In short, the molecular structure followed by refraction and susceptibility measurements and Q will throw light on dosage, toxicity of a drug. Thus the present investigations pave way for a new direction of approach for study of drug activity without recourse to techniques involving highly expensive instrumentation and highly theoretical approaches involving quantum mechanical methods.     

Structure of a monomeric mutant of the HIV-1 capsid protein

The capsid protein (CA) of HIV-1 plays a significant role in the assembly of the immature virion and is the critical building block of its mature capsid. Thus, there has been significant interest in the CA protein as a target in the design of inhibitors of early and late stage events in the HIV-1 replication cycle. However, because of its inherent flexibility from the interdomain linker and the monomer-dimer equilibrium in solution, the HIV-1 wild-type CA monomer has defied structural determinations by X-ray crystallography and nuclear magnetic resonance spectroscopy. Here we report the detailed solution structure of full-length HIV-1 CA using a monomeric mutant that, though noninfective, preserves many of the critical properties of the wild-type protein. The structure shows independently folded N-terminal (NTD) and C-terminal domains (CTD) joined by a flexible linker. The CTD shows some differences from that of the dimeric wild-type CTD structures. This study provides insights into the molecular mechanism of the wild-type CA dimerization critical for capsid assembly. The monomeric mutant allows investigation of interactions of CA with human cellular proteins exploited by HIV-1, directly in solution without the complications associated with the monomer-dimer equilibrium of the wild-type protein. This structure also permits the design of inhibitors directed at a novel target, viz., interdomain flexibility, as well as inhibitors that target multiple interdomain interactions critical for assembly and interactions of CA with host cellular proteins that play significant roles within the replication cycle of HIV-1.