Complementary functions of the Saccharomyces cerevisiae Rad2 family nucleases in Okazaki fragment maturation,mutation avoidance,and chromosome stability

Rad2 family nucleases, identified by sequence similarity within their catalytic domains, function in multiple pathways of DNA metabolism. Three members of the Saccharomyces cerevisiae Rad2 family, Rad2, Rad27, and exonuclease 1 (Exo1), exhibit both 5' exonuclease and flap endonuclease activities. Deletion of RAD27 results in defective Okazaki fragment maturation, DNA repair, and subsequent defects in mutation avoidance and chromosomal stability. However, strains lacking Rad27 are viable. The expression profile of EXO1 during the cell cycle is similar to that of RAD27 and other genes encoding proteins that function in DNA replication and repair, suggesting Exo1 may function as a back up nuclease for Rad27 in DNA replication. We show that overexpression of EXO1 suppresses multiple rad27 null mutation-associated phenotypes derived from DNA replication defects, including temperature sensitivity, Okazaki fragment accumulation, the rate of minichromosome loss, and an elevated mutation frequency. While generally similar findings were observed with RAD2, overexpression of RAD2, but not EXO1, suppressed the MMS sensitivity of the rad27 null mutant cells. This suggests that Rad2 can uniquely complement Rad27 in base excision repair (BER). Furthermore, Rad2 and Exo1 complemented the mutator phenotypes and cell cycle defects of rad27 mutant strains to differing extents, suggesting distinct in vivo nucleic acid substrates.     

Human responses to propionic acid. I. Quantification of within- and between-participant variation in perception by normosmics and anosmics

The objective of this study was to fully characterize normosmic perception of stimuli expected to cause widely varying degrees of olfactory and nasal trigeminal stimulation and to directly evaluate the possible role of olfactory nerve stimulation in nasal irritation sensitivity. During each of four identical test sessions, four anosmic and 31 normosmic participants were presented with a range of concentrations extending from peri-threshold for normosmics to supra- threshold for anosmics. For each session, odor (O) and nasal irritation (NI) sensitivities were summarized in terms of the concentrations required to produce four sensation levels ('iso-response' concentrations). Within-participant variation in these iso-response concentrations was < 10-fold for 95% of normosmics, for both O and NI. For O but not NI, these apparent fluctuations in sensitivity were largely accounted for by the uncertainty surrounding the iso-response concentrations calculated for each session. Anosmics exhibited minimal within- and between-participant variation in NI and required, for all but the highest perceptual level, a higher concentration than almost all normosmics. Between-participant variation, expressed in terms of 90% confidence interval widths, was approximately 0.5 log units for both O and NI for the highest perceptual level, but increased to approximately 0.8 and 1.8 log units, respectively, for the lowest (peri- threshold) level. Our findings suggest that: (i) most apparent variation over time in O sensitivity is actually a reflection of the uncertainty surrounding estimates of sensitivity obtained for each session; (ii) within- and between-participant variation in O sensitivity is far less than is commonly reported; and (iii) low to moderate levels of NI in normosmics are the result of relatively weak trigeminal stimulation combined with much greater olfactory activation.      

The Rapid Uptake and Release of [3H]Adenosine by Rat Cerebral Cortical Synaptosomes

Abstract: Adenosine, a putative inhibitory transmitter or modulator in the brain, is rapidly transported by rat cerebral cortical synaptosomes. The uptake may represent a facilitated diffusion process, which is saturable and temperature-dependent. In this study, the uptake process was very rapid, reaching completion within 60 s of incubation at 37°C, and had an apparent K_m value of 0.9μM and a V_max value of 5.26 pmol/mg protein/ 30 s. Over 70% of the adenosine taken up remained unchanged, whereas 14% was metabolized to inosine. Twelve percent of the adenosine was converted to nucleotides. Rapid uptake of adenosine into rat cerebral cortical synaptosomes was partially inhibited by replacing Na⁺ with choline chloride in the medium. Ca²⁺ ion is important for the uptake process, as inhibition of adenosine uptake occurs in the presence of either Co²- or EGTA. Rapid uptake of adenosine is apparently mediated by a nucleoside carrier, a conclusion based on its inhibition by a variety of purine and pyrimidine nucleosides. Uptake was inhibited by dipyridamole, hexobendine, papaverine, flurazepam, and morphine. Over 60% of the adenosine taken up by the rapid uptake system (30 s) was released by depolarizing agents. In contrast, only 30% of the adenosine taken up during a 15-min incubation period was released under the same conditions. [³H]Adenosine was the predominant purine released in the presence or absence of depolarizing agents. The basal and KCl-evoked release mechanisms were found to be at least partially Ca²⁺-dependent, however, the release of adenosine by veratridine was increased in the presence of EGTA. This finding is in agreement with the reported Ca²⁺-independent release of ATP from brain synaptosomes. The present findings suggest that there are at least two functional pools of adenosine in synaptosomes. Adenosine taken up by different uptake systems may be destined for different uses (metabolism or release) in the neuron.     

Oxypurinol-Enhanced Postischemic Recovery of the Rat Brain Involves Preservation of Adenine Nucleotides

Abstract: The present study investigated the effect of the administration of oxypurinol (40 mg/kg), an inhibitor of xanthine oxidase, on adenosine and adenine nucleotide levels in the rat brain during ischemia and reperfusion. The brains of the animals were microwaved before, at the end of a 20-min period of cerebral ischemia, and after 5, 10, 45, and 90 min of reperfusion. Cerebral ischemia was elicited by four-vessel occlusion with arterial hypotension to 45–50 mm Hg. Adenosine and adenine nucleotide levels in the oxypurinol-pretreated (administered intravenously 20 min before ischemia) rats were compared with those in nontreated animals exposed to the same periods of ischemia and reperfusion. Oxypurinol administration resulted in significantly elevated ATP levels at the end of ischemia and 5 min after ischemia, but not at 10 min after ischemia. ADP levels were also elevated, in comparison with those in the control rats, at the end of the ischemic period. Conversely, AMP levels were significantly reduced at the end of ischemia and during the initial (5 min) period of reperfusion. Adenosine levels were lower in oxypurinol-treated rats, during ischemia, and in the initial reperfusion phase. Oxypurinol administration resulted in a significant increase in the energy charge both during ischemia and after 5 min of reperfusion. Physiological indices, namely, time to recovery of mean arterial blood pressure and time to onset of respiration, were also shortened in the oxypurinol-treated animals. These beneficial effects of oxypurinol may have been a result of its purine-sparing (salvage) effects and of its ability to inhibit free radical formation by the enzyme xanthine oxidase. Preservation of high-energy phosphates during ischemia likely contributes to the cerebroprotective potency of oxypurinol.     

Distribution and release of adenosine triphosphate in rat brain

The steady-state level of brain ATP was measured after the tissue had been treated with a focused microwave irradiation system. The brain ATP content (1.50 nmol/mg tissue) obtained by microwave fixation is similar to that observed by others using fast-freezing and microwave fixation techniques. The concentrations of ATP in different brain regions show a rather uniform distribution, ranging from 1.918±0.059 (brainstem) to 2.393±0.19 (caudate) nmol/mg tissue; however, insufficient microwave fixation time seems to produce a greater regional variation of ATP. Release of ATP was investigated by placing a cup on the sensory-motor cortex. The rate for basal release of ATP is 1.43±0.14 femtomole/min/mm². A 30-fold increase in ATP release was obtained by direct stimulation of the cortex with 5 mA pulses of 0.2 msec duration at a rate of 20/sec over a period of 10 min. These results, in conjunction with others describing the potent pharmacological action of the nucleotide, seem to suggest that ATP could be a mediator in a purinergic system in the CNS.     

The actions of motilin, luteinizing hormone releasing hormone, cholecystokinin, somatostatin, vasoactive intestinal peptide, and other peptides on rat cerebral cortical neurons

The effects of a number of neuronally localized peptides have been ascertained on corticospinal and other unidentified neurons in the rat cerebral cortex. Motilin, somatostatin, and luteinizing hormone releasing hormone excited most of the corticospinal neurons on which they were tested. Cholecystokinin. Met-enkephalin, vasoactive intestinal peptide, and neurotensin also excited some corticospinal neurons. Many nonidentified neurons were excited by all of these peptides. Met-enkephalin had a depressant action on some (14%) corticospinal neurons. Leu-enkaphalin depressed many identified and nonidentified neurons and had an excitatory action on a few neurons. Both excitatory and inhibitory actions of the enkephalins were antagonized by naloxone. Thyrotropin-releasing hormone had predominantly depressant actions on the spontaneous firing of corticospinal and nonidentified neurons but did excite some unidentified cortical neurons. Secretin had no effect on the firing of most of the neurons tested.     

Monoclonal antibody 91.9H raised against sulfated mucins is specific for the 3'-sulfated Lewisa tetrasaccharide sequence

The IgG1hybridoma antibody, 91.9H, was originally raised against sulfated mucins isolated from normal human colonic mucosa. Previous studies have shown that the 91.9H antigen is expressed on normal colonic epithelial cells and the sulfomucins that they produce, but not in the normal small intestine and stomach. Tissue-specific changes occur in 91.9H antigen expression in disease: the antigen diminishes in colonic carcinomas, whereas in regions of gastric mucosa showing intestinal metaplasia and in gastric carcinomas, the antigen is expressed as a "neo-antigen." This report is concerned with elucidation, by the neoglycolipid technology, of the determinant recognized by antibody 91.9H using sulfated and sialyl oligosaccharides of Lewisa(Lea) and Lextypes, and analogs that lack sulfate, sialic acid, or fucose. Binding experiments with the lipid-linked oligosaccharides immobilized on chromatograms or on microwells, and inhibition of binding experiments with free oligosaccharides based on di-, tri- and tetrasaccharide backbones, show that the 91.9H antigenic determinant is based on a trisaccharide backbone, and consists of the 3'-sulfated Leatetrasaccharide sequence, which is a potent ligand for the E- and L-selectins. The antibody gives a relatively low signal with the 3'-sulfated non-fucosylated backbone, and has no detectable cross- reaction with the 3'-sulfated Lexisomer, nor with sialyl-Leaand - Lexanalogues. Antibody 91.9H is a valuable addition, therefore, to the repertoire of reagents for mapping details of the distribution, and determining the relative importance of sulfated and sialyl oligosaccharides as ligands for the selectins, in normal and pathological epithelia and endothelia.      

Phylogenetic utility of elongation factor-1 alpha in noctuoidea (Insecta: Lepidoptera): the limits of synonymous substitution

To test its phylogenetic utility, nucleotide sequence variation in a 1,240-bp fragment of the elongation factor-1 alpha (EF-1 alpha) gene was examined in 49 moth species representing the major groups of the superfamily Noctuoidea. Both parsimony and distance analyses supported the monophyly of nearly all groups for which there are clear morphological synapomorphies. Clades of subfamily rank and lower, probably mid-Tertiary and younger, were strongly supported. The third codon position contains 88% of variable sites, and approaches saturation at approximately 20% sequence divergence, possibly due to among-site rate heterogeneity and composition bias; higher divergences occur only in association with shifts in composition. Surprisingly, the few nonsynonymous changes appear no more phylogenetically reliable than synonymous changes. Signal strength for basal divergences is weak and fails to improve with character weighting; thus, dense taxon sampling is probably needed for strong inference from EF-1 alpha regarding deeper splits in Noctuoidea (probably early Tertiary). EF-1 alpha synonymous changes show promise for phylogeny reconstruction within Noctuidae and other groups of Tertiary age.      

Hippocalcin in the olfactory epithelium: a mediator of second messenger signaling

Intracellular Ca2+ plays an important role in a variety of second messenger cascades. The function of Ca2+ is mediated, in part, by Ca2+-binding proteins such as calmodulin, calretinin, calbindin, neurocalcin, recoverin, and visinin-like proteins (VILIPs). These proteins are highly expressed in rat olfactory receptor neurons (ORNs) and are localized to distinct intracellular regions. In the present study, we have identified another Ca2+-binding protein, hippocalcin, in the rat olfactory epithelium (OE). Olfactory/brain hippocalcin shows high sequence homology with hippocalcins expressed in mice and humans. Hippocalcin was predominantly localized to the olfactory cilia, the site of the initial events of olfactory signal transduction, and was found to regulate the activity of ciliary adenylate cyclases (ACs) and particulate guanylyl cyclases (GCs) in a Ca2+-dependent manner. These data indicate that hippocalcin is expressed in rat ORNs, and is likely to regulate second messenger cascades in a Ca2+-dependent manner.