Effect of the first window of ischemic preconditioning on mitochondrial dysfunction following global cerebral ischemia

Rats may develop sustained tolerance against lethal cerebral ischemia after exposure to a sublethal ischemic insult (ischemic preconditioning (IPC)). Two windows for the induction of tolerance by IPC have been proposed, one that occurs within 1h following IPC, and the other one that occurs 1-3 days after IPC. An important difference between these two windows is that in contrast to the second window, neuroprotection against lethal ischemia is transient in the first window. We tested the hypothesis that rapid IPC would reduce or prevent ischemia-induced changes in mitochondrial function. IPC and ischemia were produced by bilateral carotid occlusions and systemic hypotension (50 mmHg) for 2 and 10 min, respectively. The non-synaptosomal mitochondria were harvested 30 min following the 10 min 'test' ischemia. Mitochondrial rate of respiration decreased by 10% when the substrates were pyruvate and malate, and 29% when the substrates were ascorbic acid and N,N,N',N'-tetramethyl-p-phenylenediamine ( P< 0.01). The activities of complex I-III decreased in ischemic group by 16, 23 (P < 0.05) and 24%, respectively. IPC was unable to prevent decreases in the rate of respiration and activities of different complexes. These data suggest that rapidly induced IPC is unable to protect the integrity of mitochondrial oxidative phosphorylation following cerebral ischemia, perhaps explaining why IPC only provides transitory protection in the 'first window'.     

E2F and Ras synergize in transcriptionally activating p14ARF expression

The INK4a/ARF locus, which is frequently inactivated in human tumors, encodes two distinct tumor suppressive proteins, ARF and p16INK4a. ARF stabilizes and activates p53 by negating the effects of mdm2 on p53. Furthermore, its function is not restricted to the p53 pathway and it also inhibits cell proliferation in cells lacking p53. Expression of ARF is up-regulated in response to a number of oncogenic stimuli including E2F1. We show here that while oncogenic Ras does not significantly affect p1(4AR)F expression in normal human cells it activates p1(4AR)F in cells containing deregulated E2F. Moreover, oncogenic Ras and E2F1 synergize in activating p1(4AR)F expression. Activation of p1(4AR)F promoter by E2F1 persists in the absence of the consensus E2F-binding sites in this promoter, indicating that this activation also occurs through non- canonical binding sites. The activation by oncogenic Ras requires both E2F and Sp-1 activity, demonstrating the complex regulation of p14(ARF) in response to oncogenic stimuli.     

Genetic interaction between integrins and moleskin,a gene encoding a Drosophila homolog of importin-7

The Drosophila PS1 and PS2 integrins are required to maintain the connection between the dorsal and ventral wing epithelia. If alphaPS subunits are inappropriately expressed during early pupariation, the epithelia separate, causing a wing blister. Two lines of evidence indicate that this apparent loss-of-function phenotype is not a dominant negative effect, but is due to inappropriate expression of functional integrins: wing blisters are not generated efficiently by misexpression of loss-of-function alphaPS2 subunits with mutations that inhibit ligand binding, and gain-of-function, hyperactivated mutant alphaPS2 proteins cause blistering at expression levels well below those required by wild-type proteins. A genetic screen for dominant suppressors of wing blisters generated null alleles of a gene named moleskin, which encodes the protein DIM-7. DIM-7, a Drosophila homolog of vertebrate importin-7, has recently been shown to bind the SHP-2 tyrosine phosphatase homolog Corkscrew and to be important in the nuclear translocation of activated D-ERK. Consistent with this latter finding, homozygous mutant clones of moleskin fail to grow in the wing. Genetic tests suggest that the moleskin suppression of wing blisters is not directly related to inhibition of D-ERK nuclear import. These data are discussed with respect to the possible regulation of integrin function by cytoplasmic ERK.     

Specification of the direction of adhesive signaling by the integrin beta cytoplasmic domain

Integrin adhesion receptors can signal in two directions: first, they can regulate cellular behaviors by modulating cellular signaling enzymes ("outside-in signaling"); second, cells can regulate the affinity of integrins ("inside-out signaling") by such pathways. Integrin beta cytoplasmic domains (tails) mediate both types of signaling, and Src family kinases (SFKs) and talin, which bind to beta tails, are important for integrin signaling. Here, we utilized "homology scanning" mutagenesis to identify beta tail mutants selectively defective in c-Src binding and found that amino acid exchanges affecting a combination of an Arg and Thr residue in the integrin beta3 tail control the binding specificity for SFKs but have no effect on talin binding. Using beta tail mutants at these residues, we found that SFK binding to integrin beta tails is dispensable for inside-out signaling but is obligatory for cell spreading, a marker of outside-in signaling. Conversely, we found that point mutations that disrupt talin binding abolish integrin activation, but they do not inhibit SFK binding to the beta3 tail or the initiation of outside-in signaling once the integrins are in a high affinity form. Thus, we show that inside-out and outside-in integrin signaling are mediated by distinct and separable interactions of the integrin beta tails. Furthermore, based on our results, it is possible to discern the relative contributions of the direction of integrin signaling on biological functions in cell culture and, ultimately, in vivo.     

Derivation of human embryonic stem cells with NEMO deficiency

Deficiency of the nuclear factor-kappa-B essential modulator (NEMO) is a rare X-linked disorder that presents in boys as hypohydrotic ectodermal dysplasia with immunodeficiency due to defective nuclear factor-κB activation. Here we report on the generation of 2 human embryonic stem cell lines from discarded in vitro fertilization (IVF) embryos ascertained via preimplantation genetic diagnosis. We have derived two human embryonic stem cell lines that carry a T458G hypomorphic mutation in exon 4 of the NEMO (or IKBKG) gene. One of the lines is diploid male; the other is diploid female but has clonally inactivated the X-chromosome that harbors the wild-type IKBKG gene. We show that both lines are pluripotent, have the capacity to differentiate into hematopoietic progenitors, and have defective inhibitor of nuclear factor kappa-B kinase activity. These NEMO deficiency hES cell lines provide an unlimited source for differentiated cell types and may serve as a unique tool to study NEMO deficiency and potentially lead to the development of new therapies for this disease.     

Host‐seeking activity and avian host preferences of mosquitoes associated with West Nile virus transmission in the northeastern U.S.A.

Mosquito host‐seeking activity was studied using a custom‐designed trap to explore: (1) at which time interval of the night adult mosquito abatement would be most effective, and (2) if there exists an avian‐specific host‐seeking preference. Overnight trials using traps baited with dry ice showed that Aedes taeniorhynchus (Wiedemann) was most active at dusk and was then captured throughout the night. In contrast, Culex spp. (Cx. pipiens (Linnaeus) and Cx. restuans (Theobald) delayed most activity until about two h after dusk and were then captured through the night. This pattern suggests that management activities directed at adult Culex spp. would be most effective if initiated well after sunset. Mosquito capture rates in traps baited with birds in net bags were significantly greater than those with empty net bags, indicating that mosquitoes were attracted to the birds and not incidentally being sucked in by the custom trap's strong fan motor (Wilcoxon matched‐pairs signed‐ranks test, n= 24, t= 30, p < 0.05). Regression analysis showed that bird weight influenced mosquito attraction (r²= 0.21, p= 0.02). Trials with paired traps that contained different native bird species showed that Gray Catbirds, Dumatella carolinensis, attracted more mosquitoes than the heavier Northern Cardinals, Cardinalis cardinalis (paired samples t‐test, t= 2.58, df= 7, p= 0.04). However, attractiveness did not differ substantially among bird species, and Gray Catbirds did not attract more mosquitoes than all other birds combined as a group. American Robins, Turdus migratorius (n = 4) were comparable in attractiveness to other bird species, but not enough American Robins were captured for a comprehensive study of mosquito avian preference.     

Adenovirus DNA-binding protein in cells infected with wild-type 5 adenovirus and two DNA-minus, temperature-sensitive mutants, H5ts125 and H5ts149.

Studies have been done to characterize further H5ts125, an adenovirus type 5 conditionally lethal, temperature-sensitive (ts) mutant defective in initiation of DNA synthesis and to investigate whether the single-strand-specific DNA-binding (72,000 molecular weight) protein is coded by the mutated viral gene. When H5ts125-infected cells were labeled with [35S]methionine at 32 degrees C and then incubated without isotope at 39.5 degrees C, the mutant's nonpermissive temperature, the 72,000 molecular weight polypeptide was progressively degraded. Immunofluorescence examination of cells infected with wild-type virus, H5ts125, and H5ts149 (a second, unique DNA-minus mutant) showed that immunologically reactive DNA-binding protein was barely detectable in H5ts125-infected cells at 39.5 degrees C, whereas this protein was present in wild-type- and H5TS149-infected cells, that the protein made at 32 degrees C in H5ts125-infected cells lost its ability to bind specific DNA-binding protein antibody when the infected cells were shifted to 39.5 degrees C, and that if H5ts125-infected cells were shifted from the restrictive temperature to 32 degrees C, even in the presence of cycloheximide to stop protein synthesis, immunologically reactive DNA-binding protein reappeared.     

Activity,movements, and sociality of newborn Mongolian gazelle calves in the Eastern Steppe

We hand-captured, fitted with motion-sensitive VHF transmitters, and monitored 19 newborn (1–2 days old) Mongolian gazelle Procapra gutturosa Pallas, 1777 calves in Dornod, Mongolia during the 2000 and 2003 calving seasons to identify changes in activity, movements, and sociality with age. Overall, activity was highly variable throughout the day, regardless of age or year. Calf activity increased, however, from day 2 (age = 2 days; 18% total activity) to day 7 (29%), and 3-fold by day 25 (54%). By days 5–8, calves had moved an average of 6.6 km (range: 2–21 km) from capture sites but were still only seen alone or with their mother. By day 24–26, however, they were located an average of 41 km (range: 24–63 km) away from their capture site, and most (7 of 9) were in aggregations of >1000 animals. Mongolian gazelles do not display behaviour associated with “hiders” after about 2–3 weeks. This reflects the wide-ranging, nomadic existence of the species.