The mobility of an HIV-1 integrase active site loop is correlated with catalytic activity.

Replication of HIV-1 requires the covalent integration of the viral cDNA into the host chromosomal DNA directed by the virus-encoded integrase protein. Here we explore the importance of a protein surface loop near the integrase active site using protein engineering and X-ray crystallography. We have redetermined the structure of the integrase catalytic domain (residues 50-212) using an independent phase set at 1.7 A resolution. The structure extends helix alpha4 on its N-terminal side (residues 149-154), thus defining the position of the three conserved active site residues. Evident in this and in previous structures is a conformationally flexible loop composed of residues 141-148. To probe the role of flexibility in this loop, we replaced Gly 140 and Gly 149, residues that appear to act as conformational hinges, with Ala residues. X-ray structures of the catalytic domain mutants G149A and G140A/G149A show further rigidity of alpha4 and the adjoining loop. Activity assays in vitro revealed that these mutants are impaired in catalysis. The DNA binding affinity, however, is minimally affected by these mutants as assayed by UV cross-linking. We propose that the conformational flexibility of this active site loop is important for a postbinding catalytic step.     

Development of steroidogenic activity in the ovary of the prepubertal hamster: II. Production of steroids from steroidal precursors and response in vitro to cyclic adenosine monophosphate and luteinizing hormone

In vitro exposure for 2 h to 250 ng/ml of pregnenolone led to increased production of progesterone and 17 alpha-hydroxyprogesterone (17 alpha-OHP) by hamster ovaries on Days 5, 10 and 15 of age. Similar incubations with 250 ng/ml progesterone or androstenedione caused significant increases in 17 alpha-OHP or testosterone, respectively. When testosterone was added in doses of 32.5, 250 and 500 ng/ml to ovaries on Days 5-30, as early as Day 5 the ovaries aromatized the androgen to estradiol. Day 30 ovaries were the most efficient in the conversion because antral follicles, the principal site for aromatization, were then present. In terms of progesterone production, 400 ng/ml of luteinizing hormone (LH) during 4 h of in vitro incubation stimulated ovaries on Days 5, 10 and 15. Cyclic adenosine 3':5' monophosphate (cAMP) at a dose of 1 mM and 5 mM stimulated progesterone production by Days 5 and 10 ovaries more efficiently than LH. However, Day 15 ovaries produced more progesterone in response to LH compared to cAMP. These experiments establish that the steroidogenic enzymes differentiate at a very early age in the hamster ovary, even before the appearance of gonadotropin receptors. The inability of the early postnatal ovary to produce steroids is apparently attributable to lack of precursors such as cholesterol or cholesterol side chain cleavage enzymes.     

Synchronization of cell death in a dinoflagellate population is mediated by an excreted thiol protease

Regulated programmed cell death (PCD) processes have been documented in several phytoplankton species and are hypothesized to play a role in population dynamics. However, the mechanisms leading to the coordinated collapse of phytoplankton blooms are poorly understood. We showed that the collapse of the annual bloom of Peridinium gatunense, an abundant dinoflagellate in Lake Kinneret, Israel, is initiated by CO2 limitation followed by oxidative stress that triggers a PCD-like cascade. We provide evidences that a protease excreted by senescing P. gatunense cells sensitizes younger cells to oxidative stress and may consequently trigger synchronized cell death of the population. Ageing of the P. gatunense cultures was characterized by a remarkable rise in DNA fragmentation and enhanced sensitivity to H2O2. Exposure of logarithmic phase (young) cultures to conditioning media from stationary phase (old) cells sensitized them to H2O2 and led to premature massive cell death. We detected the induction of specific extracellular protease activity, leupeptin-sensitive, in ageing cultures and in lake waters during the succession of the P. gatunense bloom. Partial purification of the conditioned media revealed that this protease activity is responsible for the higher susceptibility of young cells to oxidative stress. Inhibition of the protease activity lowered the sensitivity to oxidative stress, whereas application of papain to logarithmic phase P. gatunense cultures mimicked the effect of the spent media and enhanced cell death. We propose a novel mechanistic framework by which a population of unicellular phytoplankton orchestrates a coordinated response to stress, thereby determine the fate of its individuals.     

Full-thickness skin wound explants in tissue culture: a mechanical evaluation of healing.

This study was designed to evaluate biomechanically defined wound healing in full-thickness skin explants in tissue culture. The requirement for preculture incubation of wounds in situ was characterized. Full-thickness skin incisions were made in 44 rats and closed immediately. Wounds were incubated in situ for 0, 12, 24, 36, 48, 72, or 96 hours before harvesting and placement into tissue culture media for 6 weeks. Healing was evaluated by biomechanical criteria: tensiometric distraction to wound rupture generated true stress and energy absorption data. Burst-strength (maximum true stress) and toughness (energy absorption) were five times higher in the 48-hour group than in any other group; other groups were not different from each other. This study demonstrates long-term survival of full-thickness skin in culture and shows that full-thickness skin explants heal in tissue culture. Possible explanations for the narrow window of opportunity for harvest (48 hours, no more and no less) are discussed.     

TBP flanking sequences: asymmetry of binding, long-range effects and consensus sequences

We carried out in vitro selection experiments to systematically probe the effects of TATA-box flanking sequences on its interaction with the TATA-box binding protein (TBP). This study validates our previous hypothesis that the effect of the flanking sequences on TBP/TATA-box interactions is much more significant when the TATA box has a context-dependent DNA structure. Several interesting observations, with implications for protein–DNA interactions in general, came out of this study. (i) Selected sequences are selection-method specific and TATA-box dependent. (ii) The variability in binding stability as a function of the flanking sequences for (T-A)₄ boxes is as large as the variability in binding stability as a function of the core TATA box itself. Thus, for (T-A)₄ boxes the flanking sequences completely dominate and determine the binding interaction. (iii) Binding stabilities of all but one of the individual selected sequences of the (T-A)₄form is significantly higher than that of their mononucleotide-based consensus sequence. (iv) Even though the (T-A)₄ sequence is symmetric the flanking sequence pattern is asymmetric. We propose that the plasticity of (T-A)_n sequences increases the number of conformationally distinct TATA boxes without the need to extent the TBP contact region beyond the eight-base-pair long TATA box.     

Diamidinium and iminium aromatics as new aggregators of the bacterial signaling molecule, c-di-GMP

C-di-GMP has emerged as an important bacterial signaling molecule that is involved in biofilm formation. Small molecules that can form biologically inactive complexes with c-di-GMP have the potential to be used as anti-biofilm agents. Herein, we report that water-soluble diamidinium/iminium aromatics (such as berenil), which are traditionally considered as minor groove binders of nucleic acids, are capable of aggregating c-di-GMP into G-quadruplexes via π-stacking interactions.     

Role of microorganisms in the evolution of animals and plants: the hologenome theory of evolution

We present here the hologenome theory of evolution, which considers the holobiont (the animal or plant with all of its associated microorganisms) as a unit of selection in evolution. The hologenome is defined as the sum of the genetic information of the host and its microbiota. The theory is based on four generalizations: (1) All animals and plants establish symbiotic relationships with microorganisms. (2) Symbiotic microorganisms are transmitted between generations. (3) The association between host and symbionts affects the fitness of the holobiont within its environment. (4) Variation in the hologenome can be brought about by changes in either the host or the microbiota genomes; under environmental stress, the symbiotic microbial community can change rapidly. These points taken together suggest that the genetic wealth of diverse microbial symbionts can play an important role both in adaptation and in evolution of higher organisms. During periods of rapid changes in the environment, the diverse microbial symbiont community can aid the holobiont in surviving, multiplying and buying the time necessary for the host genome to evolve. The distinguishing feature of the hologenome theory is that it considers all of the diverse microbiota associated with the animal or the plant as part of the evolving holobiont. Thus, the hologenome theory fits within the framework of the 'superorganism' proposed by Wilson and Sober.