Calculation of the solution properties of flexible macromolecules: methods and applications

While the prediction of hydrodynamic properties of rigid particles is nowadays feasible using simple and efficient computer programs, the calculation of such properties and, in general, the dynamic behavior of flexible macromolecules has not reached a similar situation. Although the theories are available, usually the computational work is done using solutions specific for each problem. We intend to develop computer programs that would greatly facilitate the task of predicting solution behavior of flexible macromolecules. In this paper, we first present an overview of the two approaches that are most practical: the Monte Carlo rigid-body treatment, and the Brownian dynamics simulation technique. The Monte Carlo procedure is based on the calculation of properties for instantaneous conformations of the macromolecule that are regarded as if they were instantaneously rigid. We describe how a Monte Carlo program can be interfaced to the programs in the HYDRO suite for rigid particles, and provide an example of such calculation, for a hypothetical particle: a protein with two domains connected by a flexible linker. We also describe briefly the essentials of Brownian dynamics, and propose a general mechanical model that includes several kinds of intramolecular interactions, such as bending, internal rotation, excluded volume effects, etc. We provide an example of the application of this methodology to the dynamics of a semiflexible, wormlike DNA.     

Acute cold exposure,leptin, and somatostatin analog (octreotide) modulate thyroid 5'-deiodinase activity

We investigated the effect of acute cold exposure, leptin, and the somatostatin analog octreotide (OCT) on thyroid type I (D1) and II (D2) deiodinase activities. Microsomal D1 and D2 activities were measured by the release of (125)I from (125)I-reverse triiodothyronine (rT(3)) under different assay conditions. Rats exposed to 4 degrees C (15, 30, 60, and 120 min) showed progressive reduction in thyroidal D1 and D2, reaching approximately 40% at 2 h (P < 0.05) despite increased circulating TSH (P < 0,05) associated with the higher thyroid D1 and D2 in hypothyroid rats. A single injection of leptin (8 microg/100 g body wt sc) induced increased thyroid and liver D1 (P < 0.05), but not thyroid D2, activities at 30 and 120 min, independently of the serum TSH rise shown only at 2 h. OCT (1 microg/kg body wt sc) increased D1 and D2 activity significantly 24 h after a single injection, with no changes in serum TSH. Therefore, leptin and somatostatin are potential physiological upregulators of thyroid deiodinases, and their low secretion during acute cold exposure may be a potential mechanism contributing to cold-induced reduction in thyroid deiodinase activity.     

Detection of HBD1 peptide in peripheral blood mononuclear cell subpopulations by intracellular flow cytometry

Production of human beta-defensin1 (HBD1) in response to LPS in monocytes, myeloid dendritic cells and plasmacytoid dendritic cells (PDC) was examined. Since PDC make up only 0.1-0.5% of the peripheral blood mononuclear cell population, we developed a method to determine HBD1 peptide levels using four-color flow cytometry, which can examine several cell surface or intracellular markers at once. Coupled with intracellular flow cytometry, we determined that PDC and monocytes only made significant amounts of HBD1 when exposed to >50ng/ml LPS for 2h. This response was limited to monocytes when ultrapure LPS was used, and was inhibited in PDC by chloroquine treatment.     

Pericentric heterochromatin becomes enriched with H2A.Z during early mammalian development

Determining how chromatin is remodelled during early development, when totipotent cells begin to differentiate into specific cell types, is essential to understand how epigenetic states are established. An important mechanism by which chromatin can be remodelled is the replacement of major histones with specific histone variants. During early mammalian development H2A.Z plays an essential, but unknown, function(s). We show here that undifferentiated mouse cells of the inner cell mass lack H2A.Z, but upon differentiation H2A.Z expression is switched on. Strikingly, H2A.Z is first targeted to pericentric hetero chromatin and then to other regions of the nucleus, but is excluded from the inactive X chromosome and the nucleolus. This targeted incorporation of H2A.Z could provide a critical signal to distinguish constitutive from facultative heterochromatin. In support of this model, we demonstrate that H2A.Z can directly interact with the pericentric heterochromatin binding protein INCENP. We propose that H2A.Z functions to establish a specialized pericentric domain by assembling an architecturally distinct chromatin structure and by recruiting specific nuclear proteins.     

Patterns of female dominance in Propithecus diadema edwardsi of Ranomafana National Park, Madagascar

Many lemur species are characterized by some form of female dominance, ranging from female feeding priority to complete female dominance, although this is a rare trait in primates and other mammals. The status of the Milne-Edwards' sifaka (Propithecus diadema edwardsi), a diurnal lemur, is ambiguous. Some short-term studies have found little or no aggression. The aim of the current, long-term study was to quantify the intersexual-dominance patterns of this sifaka. The distribution, outcome, and context of aggressive interactions were studied in four groups of wild sifakas. The majority of intersexual aggressive interactions were decided, with the loser expressing submissive behavior. Intersexual aggressive interactions occurred in all social contexts, and within all social contexts the females won the vast majority (92.7-96.0%) of aggressive interactions. While aggression rates were low (0.22/hr), this evidence suggests female dominance. We propose that female dominance exists because it provides a fitness advantage to both males and females.     

P2X2 purine receptor immunoreactivity of intraganglionic laminar endings in the mouse gastrointestinal tract

The distribution of P2X(2) purine receptor subunit immunoreactivity has been investigated in the mouse gastrointestinal tract. Immunoreactivity occurred in intraganglionic laminar endings (IGLEs) associated with myenteric ganglia throughout the gastrointestinal tract. In the esophagus, IGLEs supplied every myenteric ganglion. The proportion of ganglia supplied decreased from 85% in the stomach to 10% in the ileum, and from 50% in the caecum to 15% in the distal colon. There was substantial loss of IGLEs from myenteric ganglia of all abdominal regions after bilateral subdiaphragmatic section of the vagus nerves. IGLEs in the esophagus consisted of dense clusters of punctate immunoreactive varicosities. In the stomach and duodenum they had prominent lamellar processes and irregular, but smaller, lamellae were found in other regions. Rare immunoreactive IGLEs occurred in the submucosa of the distal colon. P2X(2) receptor immunoreactivity was on the surfaces and in the cytoplasm of a minority of nerve cells in myenteric ganglia. It is concluded that P2X(2) purine receptor immunoreactivity is a feature of IGLEs in the mouse, and that P2X receptor agonists may modulate sensitivity of the IGLEs.     

Novel application of a fibrin cell block method to evaluate melanocytic cell populations

Confirming melanocytic lineage and purity is important for experiments using cultured human melanocytes. The objective of this study was to develop a simple, reliable method to evaluate and archive cultured melanocytic cells. Melanocytes were isolated from adult skin biopsies or from neonatal foreskins using standard culturing methods. Fibrin cell blocks (FCBs) were prepared from cultured cells at passages two and six. Fibrin blocks were paraffin-embedded and sectioned for immunohistochemical (CD68, Melan-A, and HMB-45) and H & E staining. Flow cytometry was performed (Melan-A) at passage six. A mixing experiment with cultured melanocytes and fibroblasts was performed and cell population purity was determined by manual counts of positively staining cells in the FCBs and by flow cytometry. The FCB method of evaluating population purity was validated experimentally and by correlation with flow cytometry results. Preparation of a FCB followed by immunohistochemical staining is an easy and inexpensive way to confirm melanocytic lineage, estimate population purity, and provide a permanent archive of cultured cells.