Sir2-independent life span extension by calorie restriction in yeast

Calorie restriction slows aging and increases life span in many organisms. In yeast, a mechanistic explanation has been proposed whereby calorie restriction slows aging by activating Sir2. Here we report the identification of a Sir2-independent pathway responsible for a majority of the longevity benefit associated with calorie restriction. Deletion of FOB1 and overexpression of SIR2 have been previously found to increase life span by reducing the levels of toxic rDNA circles in aged mother cells. We find that combining calorie restriction with either of these genetic interventions dramatically enhances longevity, resulting in the longest-lived yeast strain reported thus far. Further, calorie restriction results in a greater life span extension in cells lacking both Sir2 and Fob1 than in cells where Sir2 is present. These findings indicate that Sir2 and calorie restriction act in parallel pathways to promote longevity in yeast and, perhaps, higher eukaryotes.     

Tight junction peptide antagonists enhance neutrophil trans-endothelial chemotaxis

Occludin is an integral membrane protein within tight junctions. Previous studies suggest it functions as a sealing element, which promotes barrier in endothelial and epithelial cell layers. Here, we examine the role of occludin in neutrophil chemotaxis, using cyclic occludin peptide antagonists that incorporate a conserved occludin cell adhesion recognition (CAR) sequence. Human umbilical vein endothelial cells were pre-treated with occludin specific cyclic peptide antagonists to examine effects on neutrophil migration towards a chemotactic gradient of 10(-7) M fMLP. The spatial organization of occludin and VE-cadherin were also assessed in control and occludin peptide-treated monolayers by immunofluorescent staining. The cyclic peptide, peptide B, which contains the CAR sequence of occludin, increased neutrophil chemotaxis in a time and dose dependent manner. Scrambled sequence peptide controls and linear peptides did not. The cyclic occludin antagonist, peptide B, disorganized junctional occludin, but apparently not VE-cadherin as assessed by immunofluorescence. The correlation between diminished occludin organization and increased neutrophil trans-endothelial chemotaxis provides additional support for occludin in the maintenance of the tight junctional barrier.     

Short-term effect of zidovudine on plasma and genital human immunodeficiency virus type 1 and viral turnover in these compartments

The effect of zidovudine on plasma and genital human immunodeficiency virus type 1 (HIV-1) was determined in 42 antiretroviral-naive HIV-1-seropositive women in Nairobi. After 7 days of zidovudine treatment, HIV-1 RNA levels decreased by 0.5 to 1.1 log(10) in plasma and genital secretions. HIV-1 RNA half-life following zidovudine treatment was 4.7, 1.3, and 0.9 days in plasma, cervix, and vagina, respectively, and significantly shorter in genital secretions than in plasma (P < 0.001). Defining the short-term effect of zidovudine on plasma and genital HIV-1 is important for improving perinatal HIV-1 interventions.     

Impairment of energy metabolism in hippocampus of rats subjected to chemically-induced hyperhomocysteinemia

Homocystinuria is an inherited metabolic disease biochemically characterized by tissue accumulation of homocysteine (Hcy). Mental retardation, ischemia and other neurological features, whose mechanisms are still obscure are common symptoms in homocystinuric patients. In this work, we investigated the effect of Hcy administration in Wistar rats on some parameters of energy metabolism in the hippocampus, a cerebral structure directly involved with cognition. The parameters utilized were 14CO2 production, glucose uptake, lactate release and the activities of succinate dehydrogenase and cytochrome c oxidase (COX). Chronic hyperhomocysteinemia was induced by subcutaneous administration of Hcy twice a day from the 6th to the 28th day of life in doses previously determined in our laboratory. Control rats received saline in the same volumes. Rats were killed 12 h after the last injection. Results showed that Hcy administration significantly diminished 14CO2 production and glucose uptake, as well as succinate dehydrogenase and COX activities. It is suggested that impairment of brain energy metabolism may be related to the neurological symptoms present in homocystinuric patients.     

Exploiting features of adenovirus replication to support mammalian kinase production

Faced with the current wealth of genomic data, it is essential to have robust and reliable methods of converting DNA sequences into their functional gene products. We demonstrate here that when conditions are established that take advantage of the replication-associated virus amplification, the virus-induced shutdown of host protein synthesis as well as the activation of signalling pathways that normally occur during virus replication, adenovirus biology can be exploited to generate a potent kinase expression system. Residual virus in the protein production has always been a limitation for adenovirus systems and we describe a DNA intercalator/ultraviolet light treatment that eliminates residual adenovirus in protein preparations that has no deleterious effect on enzyme activity. The use of mammalian cells in combination with adenovirus generated a variety of active enzymes which could not be produced in Escherichia coli or baculovirus-infected insect cells. Thus, the utility of adenovirus-mediated enzyme expression as a versatile alternative to established protein production technologies is demonstrated.     

A non-proteolytic function of separase links the onset of anaphase to mitotic exit

Separase is a protease that triggers chromosome segregation at anaphase onset by cleaving cohesin, the chromosomal protein complex responsible for sister chromatid cohesion. After anaphase, cells exit from mitosis; that is, they complete downregulation of cyclin-dependent kinase activity, undergo cytokinesis and enter G1 of the next cell cycle. Here we show that separase activation at the onset of anaphase is sufficient to promote release from the nucleolus and activation of the budding yeast phosphatase, Cdc14, a key step in mitotic exit. The ability of separase to activate Cdc14 is independent of its protease function but may involve promoting phosphorylation of the Cdc14 inhibitor Net1. This novel separase function is coregulated with its proteolytic activity by the separase inhibitor securin. This helps to explain the coupling of anaphase and mitotic exit--after securin degradation at anaphase onset, separase cleaves cohesin to trigger chromosome segregation and concurrently uses a non-proteolytic mechanism to initiate mitotic exit.     

Mechanistic insights revealed through characterization of a novel chromophore in selenophosphate synthetase from Escherichia coli

The incorporation of selenium into specific proteins and tRNAs requires selenophosphate (SePO3), whose formation is catalyzed by selenophosphate synthetase. In a Mg/ATP-dependent reaction, selenophosphate synthetase catalyzes the phosphorylation of selenide to yield AMP, inorganic phosphate, and SePO3. In this report, a previously unrecognized chromophore covalently attached to selenophosphate synthetase is characterized. The UV/Vis spectrum of selenophosphate synthetase has a feature centered at 315 nm that is irreversibly destroyed by alkylation. Moreover, addition of Zn2+, which is known to inhibit selenophosphate synthetase, reversibly quenches the 315 nm absorption. Since Zn2+ is known to bind to Cys17, these data strongly suggest that this residue participates in the 315 nm absorption. Upon incubation with both Mg2+ and ATP, the lambda(max) of the chromophore shifts to 340 nm, and it is shown that the shift requires binding of nucleotide having a hydrolyzable gamma-phosphoryl group. These data indicate that either the chromophore is directly involved in phosphoryl transfer or indirectly reflects a phosphorylation-dependent conformational change in selenophosphate synthetase. This work provides the first spectroscopic handle on catalytic steps associated with SePO3 synthesis, which will be used to study the molecular structure of the chromophore and its role in the catalytic mechanism of selenophosphate synthetase.     

Listening for bats: the hearing range of the bushcricket Phaneroptera falcata for bat echolocation calls measured in the field

The hearing range of the tettigoniid Phaneropterafalcata for the echolocation calls of freely flying mouseeared bats (Myotis myotis) was determined in the field. The hearing of the insect was monitored using hook electrode recordings from an auditory interneuron, which is as sensitive as the hearing organ for frequencies above 16 kHz. The flight path of the bat relative to the insect's position was tracked by recording the echolocation calls with two microphone arrays, and calculating the bat's position from the arrival time differences of the calls at each microphone. The hearing distances ranged from 13 to 30 m. The large variability appeared both between different insects and between different bat approaches to an individual insect. The escape time of the bushcricket, calculated from the detection distance of the insect and the instantaneous flight speed of the bat, ranged from 1.5 to more than 4s. The hearing ranges of bushcrickets suggest that the insect hears the approaching bat long before the bat can detect an echo from the flying insect.