In vitro culture of tobacco protoplasts: Use of feeder techniques to support division of cells plated at low densities

Summary Tobacco protoplasts obtained from leaf mesophyll cells and suspended in agar nutrient medium divided and produced colonies only when plated at high densities, above 10⁴ cells per ml. At such densities coalescence of the expanding colonies occurred at high frequency. Nondividing X-irradiated protoplasts used as feeder cells supported division of viable protoplasts plated at densities as low as 10² cells per ml. The feeder cell technique should thus facilitate the application of screening procedures for the isolation of colonies originating from single mutated cells occurring in a suspended population.     

Observations on the Oman Shark,Iago omanensis (Triakidae), with emphasis on the morphological and cytological changes of the oviduct and yolk sac during gestation

The shark Iago omanensis (Triakidae, Selachia) is encountered in large populations in the Gulf of Aqaba, Red Sea, at depths of 150–1,500 m. It is a placental viviparous species, reproductive all year round and giving birth to four (occasionally five) young of 170- to 180-mm total length (TL). Its distribution and morphometrics, as well as histological and cytological changes in the oviducts, were studied. The ratio of weight of the female genital organs to body weight changes from 0.7% in nongravid females to 19.8% in the final stages of pregnancy. The ripe, liberated eggs, which are 11–12 mm long and 5 mm wide, pass through the nidamental gland and settle in the uterus. The embryo attains 9- to 11-mm TL and settles on a protruding ridge of the submucosa, covered with a microvillar endometrium. At this site of attachment, a placenta is formed and the participating uterine endometrium and wall of the yolk sac undergo profound histocytological changes, forming two parts of this organ. Three forms of food provisioning occur in the growing embryos: (1) lecithotrophic, based on yolk transported from the egg to the embryonic gut via the umbilical cord; (2) mixed food provision, during which, in addition to nourishment provided via the umbilicus, food is transported across the placenta through transfer from the female blood vascular system to the embryonic yolk sac via the trophic villi of the yolk sac; and (3) histotrophic, when all yolk reserves have been used and nutrition is provided from the so-called “milk” within the yolk sac, metabolized by the trophic structures of the sac and transported by blood vessels. Despite the gradual utilization of yolk, the yolk sac mass initially increases from 0.5–1.0 cc to 2.0–2.2 cc with the addition of primary and secondary trophic villi until, during the final stages of embryogenesis, it decreases again to 1.4–1.6 cc. Neonate juveniles are 35–40 times heavier than the original eggs. J. Morphol. 236:151–165, 1998. © 1998 Wiley-Liss, Inc.     

Insights into the organization of biochemical regulatory networks using graph theory analyses

Graph theory has been a valuable mathematical modeling tool to gain insights into the topological organization of biochemical networks. There are two types of insights that may be obtained by graph theory analyses. The first provides an overview of the global organization of biochemical networks; the second uses prior knowledge to place results from multivariate experiments, such as microarray data sets, in the context of known pathways and networks to infer regulation. Using graph analyses, biochemical networks are found to be scale-free and small-world, indicating that these networks contain hubs, which are proteins that interact with many other molecules. These hubs may interact with many different types of proteins at the same time and location or at different times and locations, resulting in diverse biological responses. Groups of components in networks are organized in recurring patterns termed network motifs such as feedback and feed-forward loops. Graph analysis revealed that negative feedback loops are less common and are present mostly in proximity to the membrane, whereas positive feedback loops are highly nested in an architecture that promotes dynamical stability. Cell signaling networks have multiple pathways from some input receptors and few from others. Such topology is reminiscent of a classification system. Signaling networks display a bow-tie structure indicative of funneling information from extracellular signals and then dispatching information from a few specific central intracellular signaling nexuses. These insights show that graph theory is a valuable tool for gaining an understanding of global regulatory features of biochemical networks.     

SNAVI: Desktop application for analysis and visualization of large-scale signaling networks

Background  

Studies of cellular signaling indicate that signal transduction pathways combine to form large networks of interactions. Viewing protein-protein and ligand-protein interactions as graphs (networks), where biomolecules are represented as nodes and their interactions are represented as links, is a promising approach for integrating experimental results from different sources to achieve a systematic understanding of the molecular mechanisms driving cell phenotype. The emergence of large-scale signaling networks provides an opportunity for topological statistical analysis while visualization of such networks represents a challenge.     

Anticonvulsant profile and teratogenic evaluation of potent new analogues of a valproic acid urea derivative in NMRI mice

BACKGROUND: Valproic acid (VPA) is used to treat epilepsy and bipolar disorders, as well as for migraine prophylaxis. However, its clinical use is limited by two life-threatening side effects: hepatotoxicity and teratogenicity. To develop a more potent and safer second-generation VPA drug, the urea derivatives of four VPA analogs (2-ethyl-3-methylpentanoyl urea, 2-ethylhexanoyl urea, 2-ethyl-4-methylpentanoyl urea, and 2-methylbutanoyl urea) were synthesized. METHODS: Four CNS-active analogs of a VPA urea derivative testedthe anticonvulsant activity in the maximal electroshock seizure test (MES) and subcutaneous metrazol seizure threshold test (scMet). Teratogenic effects of these compounds were evaluated in NMRI mice susceptible to VPA-induced teratogenicity by comparison with VPA. RESULTS: All four VPA analogs showed superior anticonvulsant activity over VPA. Compared with VPA, which induced neural tube defects (NTDs) in fetuses at 1.8 and 3.6 mmol/kg, the analog derivatives induced no NTDs at any concentration up to 4.8 mmol/kg (except for a single abnormality at 3.6 mmol/kg with 2-ethyl-3-methylpentanoyl urea). Skeletal examination also revealed that the acylurea derivatives induced vertebral and rib abnormalities in fetuses markedly less frequently than VPA. Our results confirmed that the analogue derivatives are significantly less teratogenic than VPA in NMRI mice. CONCLUSIONS: The CNS-active VPA analogs containing a urea moiety, which have better anticonvulsant potency and lack teratogenicity, are good potential candidates as second-generation VPA antiepileptic drugs. Birth Defects Res (Part B) 86:394–401, 2009. © 2009 Wiley-Liss, Inc.     

Rapid Sampling of Molecular Motions with Prior Information Constraints

Proteins are active, flexible machines that perform a range of different functions. Innovative experimental approaches may now provide limited partial information about conformational changes along motion pathways of proteins. There is therefore a need for computational approaches that can efficiently incorporate prior information into motion prediction schemes. In this paper, we present PathRover, a general setup designed for the integration of prior information into the motion planning algorithm of rapidly exploring random trees (RRT). Each suggested motion pathway comprises a sequence of low-energy clash-free conformations that satisfy an arbitrary number of prior information constraints. These constraints can be derived from experimental data or from expert intuition about the motion. The incorporation of prior information is very straightforward and significantly narrows down the vast search in the typically high-dimensional conformational space, leading to dramatic reduction in running time. To allow the use of state-of-the-art energy functions and conformational sampling, we have integrated this framework into Rosetta, an accurate protocol for diverse types of structural modeling. The suggested framework can serve as an effective complementary tool for molecular dynamics, Normal Mode Analysis, and other prevalent techniques for predicting motion in proteins. We applied our framework to three different model systems. We show that a limited set of experimentally motivated constraints may effectively bias the simulations toward diverse predicates in an outright fashion, from distance constraints to enforcement of loop closure. In particular, our analysis sheds light on mechanisms of protein domain swapping and on the role of different residues in the motion.     

The zebrafish as a model organism for the study of apoptosis

Apoptosis plays important roles in embryogenesis, tissue homeostasis, and immune system regulation. The zebrafish (Danio rerio) is a powerful vertebrate model organism that has been extensively used to study apoptotic cell death during normal development and under conditions of cellular stress. In the past 5 years, a detailed picture has begun to emerge of the molecular underpinnings of the cell-intrinsic and the cell-extrinsic apoptosis signaling pathways in zebrafish. We begin this review with an introduction to the techniques and experimental approaches that are used to study apoptosis in zebrafish. We follow with a general overview of developmental apoptosis during zebrafish embryogenesis. Finally, we present a comprehensive review of the intrinsic and extrinsic apoptosis pathways in zebrafish, focusing on the high degree of conservation with humans and other mammals. Recent publications that draw upon the unique advantages of the zebrafish system to study novel aspects of apoptosis regulation and function are highlighted throughout.