We present an application of an F2 screening method for recovering and estimating the frequencies of rare alleles that confer insect resistance to a transgenic corn variety producing Bacillus ...thuringiensis Berliner crystal protein toxin (Bt corn). Based on a sample of 91 female Ostrinia nubilalis (Hubner) we show with 95% confidence that the frequency of B. thuringiensis resistance alleles from a wild Minnesota population is 0.013. This is an upper limit to the estimated allele frequency and does not provide clear evidence that 1 of the assumptions of the refuge plus high-dose strategy will or will not be met. With additional sampling, a more precise estimate of resistance allele frequency could be obtained that would clearly support or refute 1 of the assumptions of the refuge plus high-dose strategy. Variable costs of the screening method were $19.70 per female line, but these could be reduced by improved collecting, rearing, and handling methods
Xenopus egg extracts which assemble replication competent nuclei in vitro were depleted of lamin B3 using monoclonal antibody L6 5D5 linked to paramagnetic beads. After depletion, the extracts were ...still capable of assembling nuclei around demembranated sperm heads. Using field emission in lens scanning electron microscopy (FEISEM) we show that most nuclei assembled in lamin B3-depleted extracts have continuous nuclear envelopes and well formed nuclear pores. However, several consistent differences were observed. Most nuclei were small and only attained diameters which were half the size of controls. In a small number of nuclei, nuclear pore baskets, normally present on the inner aspect of the nuclear envelope, appeared on its outer surface. Finally, the assembly of nuclear pores was slower in lamin B3-depleted extracts, indicating a slower overall rate of nuclear envelope assembly. The results of FEISEM were confirmed using conventional TEM thin sections, where again the majority of nuclei assembled in lamin B3-depleted extracts had well formed double unit membranes containing a high density of nuclear pores. Since nuclear envelope assembly was mostly normal but slow in these nuclei, the lamin content of 'depleted' extracts was investigated. While lamin B3 was recovered efficiently from cytosolic and membrane fractions by our procedure, a second minor lamin isoform, which has characteristics similar to those of the somatic lamin B2, remained in the extract. Thus it is likely that this lamin is necessary for nuclear envelope assembly. However, while lamin B2 did not co-precipitate with lamin B3 during immunodepletion experiments, several protein species did specifically associate with lamin B3 on paramagnetic immunobeads. The major protein species associated with lamin B3 migrated with molecular masses of 102 kDa and 57 kDa, respectively, on one-dimensional polyacrylamide gels. On two-dimensional O'Farrell gels the mobility of the 102 kDa protein was identical to the mobility of a major nuclear matrix protein, indicating a specific association between lamin B3 and other nuclear matrix proteins. Nuclei assembled in lamin B3-depleted extracts did not assemble a lamina, judged by indirect immunofluorescence, and failed to initiate semi-conservative DNA replication. However, by reinoculating depleted extracts with purified lamin B3, nuclear lamina assembly and DNA replication could both be rescued. Thus it seems likely that the inability of lamin-depleted extracts to assemble a replication competent nucleus is a direct consequence of a failure to assemble a lamina.
It has recently been reported that the hsp56 component of glucocorticoid receptor heterocomplexes is an immunophilin of the FK506 binding class Yem, A. W., Tomasselli, A. G., Heinrikson, R. L., ...Zurcher-Neely, H., Ruff, V. A., Johnson, R. A., & Deibel, M. R. (1992) J. Biol. Chem. 267, 2868-2871; Tai, P. K., Albers, M. W., Chang, H., Faber, L. E., & Schreiber, S. L. (1992) Science 256, 1315-1318. The existence of binding proteins for these two potent groups of immunosuppressants in the same molecular complex compels us to ask whether FK506 affects glucocorticoid receptor function. We show here that hsp56 is a component of the native L-cell glucocorticoid receptor heterocomplex and that 3HFK506 binds to the immunopurified, untransformed receptor complex. However, at concentrations in excess of those required to occupy all of its binding sites on hsp56, FK506 does not affect the steroid binding activity of the receptor nor does it stabilize or dissociate the receptor-hsp90 complex. FK506 does not affect steroid-mediated hsp90 dissociation from the receptor in vitro, and it does not affect steroid-mediated nuclear transfer of the receptor or steroid-mediated transcriptional enhancement from a reporter in intact cells. When immunopurified mouse glucocorticoid receptor is reconstituted into a heat shock protein complex by rabbit reticulocyte lysate, hsp56 is present in the reconstituted complex in addition to hsp90 and hsp70. FK506, however, does not affect reconstitution of the complex or return of the receptor to the steroid binding state, a change of conformation that occurs upon receptor association with hsp90.
Benzodiazepines are allosteric modulators of the GABA(A) receptor. The traditionally prescribed benzodiazepines are nonselective and suffer from numerous side effects. Upon the identification of ...receptor subtypes, we set out to discover selective agents with the anticipation that these agents would have superior therapeutic potential. Herein, we describe the synthesis and biological evaluation of substituted 7,8,9,10-tetrahydroimidazo1,2-cpyrido3,4-epyrimidin-5(6H)-ones and disclose that these compounds exhibit functional selectivity at the benzodiazepine receptor of GABA(A) receptor subtypes. The alpha(2)/alpha(3)-selective partial agonist 42 exhibited potent in vivo activity.
We sought to assess the effects of second-hand smoke (SHS) and gender on infarct size in young rats exposed in utero or in the neonatal to adolescent period, or both.
We previously demonstrated that ...exposure to SHS increases infarct size in a rat model of ischemia and reperfusion, with a dose-response relation. These results are consistent with epidemiologic studies demonstrating that SHS increases risk of death from heart disease.
Thirty-one pregnant female rats were randomly divided into two groups: those exposed to SHS and a control group (non-SHS). After 3 weeks, each rat had given birth to 10 to 12 rats. One hundred one neonatal rats were divided into four groups according to exposure to SHS in utero (SHSu) and randomized to SHS exposure in the neonatal to adolescent period (SHSna). After 12 weeks, all rats were subjected to 17 min of left coronary artery occlusion and 2 h of reperfusion.
Birth mortality was higher in the SHSu group than in the non-SHSu group (11.9% vs. 2.8%, p < 0.001). Body weight of neonatal rats at 3 and 4 weeks in the two SHSu groups was lower than that of rats in the two non-SHSu groups (p < 0.001). Exposure to SHSna increased endothelin-1 levels in plasma (p = 0.001). In all 70 young rats who survived the neonatal period, infarct size (Infarct mass/Risk area x 100%) was greater in the SHSna groups than in the non-SHSna groups (p = 0.005) and in the male groups than in the female groups (p < 0.001).
Exposure to SHS in the neonatal to adolescent period and male gender increased myocardial infarct size in a young rat model of ischemia and reperfusion. These results are consistent with epidemiologic studies demonstrating that SHS increases the health risk to neonates and adolescents.
We have identified a new pathogenic mechanism for an inherited muscular dystrophy in which functional haploinsufficiency of the extracellular matrix protein collagen VI causes Bethlem myopathy. The ...heterozygous COL6A1 mutation results in a single base deletion from the mRNA and a premature stop codon. The mutant mRNA is unstable, subject to nonsense-mediated mRNA decay, and is almost completely absent both from patient fibroblasts and skeletal muscle, resulting in haploinsufficiency of the α1(VI) subunit and reduced production of structurally normal collagen VI. This is the first example of a muscular dystrophy caused by haploinsufficiency of a structural protein or member of the dystrophin-glycoprotein complex, and identifies collagen VI as a critical contributor to cell-matrix adhesion in skeletal muscle.
Early diagnosis of perinatally transmitted human immunodeficiency virus type 1 (HIV) infection can guide early interventions. HIV coculture and DNA polymerase chain reaction (DNA-PCR) detect few ...HIV-infected infants at birth and 90%–100% by age 3 months. Because extracellular HIV RNA may appear soon after infection, a plasma HIV RNA assay was compared with DNAPCR for early detection of perinatally infected infants. Blood-draw specimens (108) obtained at the same time from 49 HIV-infected infants and 10 specimens from 8 uninfected infants were tested. HIV RNA and DNA-PCR positivity rates were 56% and 33%, respectively, in 36 specimens from 36 infants <28 days of age (binomial test, P = .001). Among 81 specimens obtained after age 14 days, 79 (98%) were positive by HIV RNA testing. No HIV-infected infant specimens were DNAPCR-positive and HIV RNA-negative. All specimens from 8 uninfected infants were HIV RNA-negative. These results suggest that plasma HIV RNA was detectable earlier and more reliably than HIV DNA in perinatal infection.
Recent studies have renewed interest in the role of limbic structures, such as the cingulate cortex, in nociception. To investigate the involvement of the limbic system in pain and temperature ...perception further, we have quantified ratings of innocuous and noxious thermal stimuli in a patient with schizoaffective disorder before and after 2 surgical procedures. Psychophysical tests were conducted at a control session prior to surgery. Postoperative test sessions were conducted up to 10 weeks after bilateral cingulotomy and for 3 months after a subsequent bilateral anterior internal capsulotomy. A contact thermal stimulator delivered ascending (39-50 degrees C) and descending (22-2 degrees C) series of stimuli to the patient's volar forearm. The patient was trained to rate the innocuous warmth and cold and the pain associated with each stimulus. A cold pressor test was used to obtain a measure of cold pain tolerance. Compared to pre-operative levels, cingulotomy/capsulotomy resulted in moderately diminished warmth perception and an elevated heat pain threshold and increased ratings to suprathreshold noxious heat stimuli (hyperpathia). Prior to surgery, the patient perceived all cold stimuli as cold but not painful. However, after cingulotomy and capsulotomy, cold stimuli were rated significantly colder and stimuli less than or equal to 12 degrees C evoked pain. Compared to normal control subjects, the patient's ratings of innocuous and noxious cold stimuli were reduced pre-operatively but elevated postoperatively and cold pain tolerance was elevated pre-operatively but reduced postoperatively. These altered ratings of noxious heat and cold stimuli were reflected on both a pain intensity and pain affect (unpleasantness) scale. In summary, these data suggest that cingulotomy and capsulotomy disinhibited the patient's noxious heat and cold appreciation. These findings provide support for a role of the cingulate cortex and frontal cortical regions in the perception of innocuous and noxious thermal stimuli and suggest that under normal conditions, these areas may act to suppress the subjective intensity of noxious heat and cold.
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