High Rates of Actin Filament Turnover in Budding Yeast and Roles for Actin in Establishment and Maintenance of Cell Polarity Revealed Using the Actin Inhibitor Latrunculin-A

We report that the actin assembly inhibitor latrunculin-A (LAT-A) causes complete disruption of the yeast actin cytoskeleton within 2–5 min, suggesting that although yeast are nonmotile, their actin filaments undergo rapid cycles of assembly and disassembly in vivo. Differences in the LAT-A sensitivities of strains carrying mutations in components of the actin cytoskeleton suggest that tropomyosin, fimbrin, capping protein, Sla2p, and Srv2p act to increase actin cytoskeleton stability, while End3p and Sla1p act to decrease stability. Identification of three LAT-A resistant actin mutants demonstrated that in vivo effects of LAT-A are due specifically to impairment of actin function and implicated a region on the three-dimensional actin structure as the LAT-A binding site.

LAT-A was used to determine which of 19 different proteins implicated in cell polarity development require actin to achieve polarized localization. Results show that at least two molecular pathways, one actindependent and the other actin-independent, underlie polarity development. The actin-dependent pathway localizes secretory vesicles and a putative vesicle docking complex to sites of cell surface growth, providing an explanation for the dependence of polarized cell surface growth on actin function. Unexpectedly, several proteins that function with actin during cell polarity development, including an unconventional myosin (Myo2p), calmodulin, and an actin-interacting protein (Bud6/Aip3p), achieved polarized localization by an actin-independent pathway, revealing interdependence among cell polarity pathways. Finally, transient actin depolymerization caused many cells to abandon one bud site or mating projection and to initiate growth at a second site. Thus, actin filaments are also required for maintenance of an axis of cell polarity.

     

Age does not alter protein kinase C isozymes mRNA expression in rat brain

Calcium and phospholipid dependent Protein kinase C (PKC) may play a role in memory function and pathogenesis of many neurodegenerative disorders such as Alzheimer's disease (AD). Abnormal phosphorylation by PKC as well as reduced levels of PKC has been implicated in the neurodegeneration associated with AD and aging. Recently, many subtypes of PKC isozymes have been identified by molecular biology techniques which are expressed differentially in various regions of the brain. The reduction and alterations in the activities and distribution of these subtypes of PKC isozymes may be accountable for the decline of selective neurons during aging. In order to investigate the role of PKC isozymes during aging, we examined the distribution of PKC-, , and mRNA, expressions between young (4 months) and old (25 months) rat brains using in situ hybridization histochemistry. Our studies showed that signals of three isoforms of PKC mRNA vary in cortical and hippocampal regions. However, no change was detected in any of the PKC isoforms mRNA expressions in aged animals.     

Monophyly of the order Rodentia inferred from mitochondrial DNA sequences of the genes for 12S rRNA, 16S rRNA, and tRNA-valine

A recent analysis of amino acid sequence data (Graur et al.) suggested that the mammalian order Rodentia is polyphyletic, in contrast to most morphological data, which support rodent monophyly. At issue is whether the hystricognath rodents, such as the guinea pig, represent an independent evolutionary lineage within mammals, separate from the sciurognath rodents. To resolve this problem, we sequenced a region (2,645 bp) of the mitochondrial genome of the guinea pig containing the complete 12S ribosomal RNA, 16S ribosomal RNA, and transfer RNA(VAL) genes for comparison with the available sciurognath and other mammalian sequences. Several methods of analysis and statistical tests of the data all show strong support for rodent monophyly (91%-98% bootstrap probability, or BP). Calibration with the mammalian fossil record suggests a Cretaceous date (107 mya) for the divergence of sciurognaths and hystricognaths. An older date (38 mya) for the controversial Mus- Rattus divergence also is supported by these data. Our neighbor-joining analyses of all available sequence data (25 genes) confirm that some individual genes support rodent polyphyly but that tandem analysis of all data does not. We propose that the conflicting results are due to several compounding factors. The unique biochemical properties of some hystricognath metabolic proteins, largely responsible for generating this controversy, may have a single explanation: a cascade effect resulting from inactivation of the zinc-binding abilities of insulin. After excluding six genes possibly affected by insulin inactivation, analyses of all available sequence data (7,117 nucleotide sites, 3,099 amino acid sites) resulted in strong support for rodent monophyly (94% BP for DNA sequences, 90% for protein sequences), which lends support to the insulin-cascade hypothesis.      

Failure to detect "cap" structures in mitochondrial DNA-coded poly(A)-containing RNA from HeLa cells.

The structure of the 5'-termini has been investigated in mitochondrial DNA-coded poly(A)-containing RNA from HeLa cells. For this purpose, mitochondrial RNA isolated from cells labeled for 3 hours with [32P]orthophosphate in the presence of 20 microgram/ml camptothecin, and selected for poly(A) content by two passages through oligo(dT)-cellulose, was digested either with the nuclease P1 or with a mixture of RNases: the digestion products were then fractionated by two-dimensional electrophoresis. No "cap" structures were detected under conditions where the presence of such structures in one out of five to ten RNA molecules would have been recognized. It is, therefore, likely that "cap" structures are completely absent in HeLa cell mitochondrial poly(A)-containing RNA.     

Antifilarial and Antibiotic Activities of Methanolic Extracts of Melaleuca cajuputi Flowers

We evaluated the activity of methanolic extracts of Melaleuca cajuputi flowers against the filarial worm Brugia pahangi and its bacterial endosymbiont Wolbachia. Anti-Wolbachia activity was measured in worms and in Aedes albopictus Aa23 cells by PCR, electron microscopy, and other biological assays. In particular, microfilarial release, worm motility, and viability were determined. M. cajuputi flower extracts were found to significantly reduce Wolbachia endosymbionts in Aa23 cells, Wolbachia surface protein, and microfilarial release, as well as the viability and motility of adult worms. Anti-Wolbachia activity was further confirmed by observation of degraded and phagocytized Wolbachia in worms treated with the flower extracts. The data provided in vitro and in vivo evidence that M. cajuputi flower extracts inhibit Wolbachia, an activity that may be exploited as an alternative strategy to treat human lymphatic filariasis.     

Past and present fish species recorded in the estuarine Lake Ichkeul,northern Tunisia

Lake Ichkeul in northern Tunisia is a Ramsar site, a MAB Biosphere Reserve and a UNESCO World Heritage Site. The system is one of the most important coastal wetlands in North Africa, especially as an over-wintering area for migratory birds, particularly Palaearctic waterfowl. The present study was aimed at diagnosing the status of fish species in Lake Ichkeul and documenting their annual and seasonal occurrence within the system. Fish samples were collected monthly at 22 sites from March 2011 to April 2012 using a variety of gears, including a dragnet, gillnet, trammelnet, frynet and beach-seine. Temperature and salinity measurements were taken at each site, while fishes were obtained from only 11 sites, representing mainly lacustrine stations. A total of 18 fish species belonging to 14 genera and 12 families were identified, with Mugilidae dominating with four species, followed by Syngnathidae with three species. This study represents a relative recovery in terms of fish species richness, since only 13 species were recorded here between 2003 and 2007. However, the numbers of fish caught, especially of the commercial taxa, seem to have declined in recent years, and the number of species currently present is much lower than that of several decades ago. Increasing human pressures, particularly reductions in catchment freshwater inputs, a breakdown in ecosystem connectivity, pollution and over-fishing appear to be the principal causes for the decline in fish abundance and diversity.     

Regulation of prostaglandin biosynthesis by nitric oxide is revealed by targeted deletion of inducible nitric-oxide synthase

We investigated the effects of targeted deletion of the inducible NO synthase (iNOS) gene on the formation of prostaglandins in vivo and ex vivo. Peritoneal macrophages were obtained from control and iNOS-deficient mice, and prostaglandin E(2) (PGE(2)) was quantified after stimulation with gamma-interferon and lipopolysaccharide to induce COX-2. Total nitrate and nitrite production was completely abolished in cells from iNOS-deficient animals compared with control cells. PGE(2) formation by cells from iNOS-deficient animals was decreased compared with cells from control animals 80% at 12 h (0.85 +/- 0.90 ng/10(6) cells versus 15.4 +/- 2.1 ng/10(6) cells, p < 0.01) and 74% at 24 h (9.4 +/- 4.3 ng/10(6) cells versus 36.8 +/- 4.1 ng/10(6) cells, p < 0.01). COX-2 protein expression was not significantly different in cells from control or knockout animals. Levels of PGE(2) in the urine of iNOS-deficient mice were decreased 78% (0.24 +/- 0.14 ng/mg of creatinine versus 1.09 +/- 0.66 ng/mg of creatinine, p < 0.01) compared with control animals. In addition, the levels of urinary F(2)-isoprostanes, an index of endogenous oxidant stress, were significantly decreased in iNOS-deficient animals. In contrast, the levels of thromboxane B(2) derived from platelets allowed to aggregate ex vivo were significantly increased in iNOS-deficient mice compared with wild-type mice. These studies support the hypothesis that NO and/or NO-derived species modulate cyclooxygenase activity and eicosanoid production in vivo.     

Effects of NMDA and ferrous sulfate on oxidation and cell death in primary neuronal cultures

Excessive oxidative radical production has been implicated in a variety of neurodegerative processes including NMDA (N-methyl-D-aspartate) mediated excitotoxicity. To determine the relationship of oxidation to NMDA-receptor mediated neuronal death, we exposed rat primary cortical neuronal cultures to ferrous sulfate and the fluorescent dyes dichlorofluorescin diacetate (H(2)DCF) and propidium iodide (PI) to monitor reactive oxygen species (ROS) and cell death, respectively in the same cultures. Ferrous sulfate (FeSO(4)) caused a dose-dependent increase in cellular oxidation with an ED(50) of approximately 136 microM. Levels of oxidation increased over time reaching maximum levels between 15 and 25 min. Ferrous sulfate (ED(50) approximately 241 microM) treatment for 25 min caused a delayed and progressive neuronal death that was comparable to NMDA (100 microM, 25 min) delayed neuronal death. NMDA (100 microM, 25 min) alone did not result in measurable increases of DCF fluorescence. However, when combined with 40 microM FeSO(4), NMDA dose-dependently increased H(2)DCF fluorescence. Despite the increase in DCF oxidation, combinations of FeSO(4) with NMDA did not synergize or accelerate NMDA-receptor mediated or glutamate-mediated excitotoxicity. Although excessive amounts FeSO(4) induced oxidation can cause delayed neuronal death, these findings suggest that oxidative stress is not the key factor in triggering the NMDA mediated excitotoxic cascade.     

The Effects of Social Experience on Aggressive Behavior in the Green Anole Lizard (Anolis carolinensis)

To understand how context-specific aggression emerges from past experience, we examined how consecutive aggressive encounters influence aggressive behavior and stress responses of male green anole lizards ( Anolis carolinensis ). Animals were shown a video clip featuring an aggressively displaying conspecific male, which provoked aggressive responding, while control animals viewed a neutral video. After 5 d of interaction with the videos, both the subject and control groups were presented with a live conspecific. As a non-invasive assay of stress responses, we measured changes in body color and eyespot darkness, two features known to be strongly correlated with titers of stress hormones. Our results demonstrate that experience increased aggression in male anoles, but that increases in aggression to a repeated stimulus were transient. Tests with a novel conspecific indicate that the experienced animals remained aggressive when presented with novel stimuli. Although there were differences in the morphological indicators of the stress response between experimental and control groups during video presentations, there were no differences when presented with novel conspecifics. These data indicate that experience-dependent differences were not mediated by differences in the 'stressfulness' of aggressive interaction, as thought to be the case for animals in chronic subordinate/dominant dyads. We suggest that habituation and reinforcement interact to promote aggressive responding and to restrict it to novel individuals. Such context specificity is a hallmark of natural patterns of aggression in territorial species.