Some experiments on the function of the labyrinth. II. Histological observations with regard to these experiments.
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Ear, Inner↗
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Xenopus aristaless-related homeobox (xARX) gene product functions as both a transcriptional activator and repressor in forebrain development.
Mutations in the aristaless-related homeobox (ARX) gene have been found in patients with a variety of X-linked mental retardation syndromes with forebrain abnormalities, including lissencephaly. Arx is expressed in the developing mouse, Xenopus, and zebrafish forebrain. We have used whole-mount in situ hybridization, overexpression, and loss-of-function studies to investigate the involvement of xArx in Xenopus brain development. We verified that xArx is expressed in the prospective diencephalon, as the forebrain is patterned and specified during neural plate stages. Expression spreads into the ventral and medial telencephalon as development proceeds through neural tube and tadpole stages. Overexpression of xArx resulted in morphological abnormalities in forebrain development, including loss of rostral midline structures, syn- or anophthalmia, dorsal displacement of the nasal organ, and ventral neural tube hyperplasia. Additionally, there is a delay in expression of many molecular markers of brain and retinal development. However, expression of some markers, dlx5 and wnt8b, was enhanced in xArx-injected embryos. Loss-of-function experiments indicated that xArx was necessary for normal forebrain development. Expansion of wnt8b expression depended on xArx function as a transcriptional repressor, whereas ectopic expression of dlx5, accompanied by development of ectopic otic structures, depended on function of Arx as a transcriptional activator. These results suggest that Arx acts as a bifunctional transcriptional regulator in brain development.
Animals↗
DREF is required for efficient growth and cell cycle progression in Drosophila imaginal discs.
Based on overexpression studies and target gene analyses, the transcription factor DNA replication-related element factor (DREF) has been proposed to regulate growth and replication in Drosophila melanogaster. Here we present loss-of-function experiments to analyze the contribution of DREF to these processes. RNA interference-mediated extinction of DREF function in vivo demonstrates a requirement for the protein for normal progression through the cell cycle and consequently for growth of imaginal discs and the derived adult organs. We show that DREF regulates the expression of genes that are required for the transition of imaginal disc cells through S phase. In conditions of suppressed apoptosis, DREF activation can cause overgrowth of developing organs. These data establish DREF as a global regulator of transcriptional programs that mediate cell proliferation and organ growth during animal development.
Animals↗
Aftereffects of the surprising presentation and omission of appetitive reinforcers on key-pecking performance in pigeons.
The reinforcement-omission effect (ROE), also known as frustration effect, refers to greater response strength immediately after nonreinforcement (N) than reinforcement (R). The ROE was traditionally interpreted as transient invigoration after N induced by primary frustration. Pigeons demonstrate similar ROEs whether outcomes are surprising (partial R) or expected (discrimination training) in runway (Experiment 1) and Skinner box situations (Experiments 2-3). Variations in the interval between N and the opportunity to respond indicate that the ROE results from an aftereffect of food consumption (Experiment 4). Increasing reinforcer magnitude increased the after-R effect, without modifying the after-N function (Experiment 5). These results are reviewed in the context of comparative research on spaced-trial successive negative contrast and related phenomena that have failed to appear in experiments involving nonmammalian vertebrates.
Animals↗
A far-upstream sequence of the wheat histone H3 promoter functions differently in rice and tobacco cultured cells.
The cis-regulatory function of a far-upstream sequence (-1,711 to -186) of the promoter of the wheat gene for histone H3 (TH012) was analyzed in cultured rice and tobacco cells in a transient expression system with the gene for beta-D-glucuronidase as a reporter gene. The far-upstream sequence was necessary for full activity of the H3 promoter in rice cells but did not enhance the activity of the proximal promoter in tobacco cells. Dissection analysis of the far-upstream sequence revealed the existence of several positive and negative cis-acting sequences in this region, some of which functioned differently in rice and tobacco cells. In gain-of-function experiments with rice cells, the sequence from -848 to -704, containing the CCAAT and octamer (CaCGGATC) motifs, functioned in an orientation-independent manner, whereas the sequence from -703 to -486 functioned in an orientation-dependent manner. By contrast, both sequences exhibited an orientation-dependent cis-function in tobacco cells. These findings suggest that some cis-regulatory sequences in the far-upstream region of the H3 promoter function differently in rice and tobacco cells.
Base Sequence↗
The basic helix-loop-helix factor olig2 is essential for the development of motoneuron and oligodendrocyte lineages.
Sonic hedgehog (Shh), an organizing signal from ventral midline structures, is essential for the induction and maintenance of many ventral cell types in the embryonic neural tube. Olig1 and Olig2 are related basic helix-loop-helix factors induced by Shh in the ventral neural tube. Although expression analyses and gain-of-function experiments suggested that these factors were involved in motoneuron and oligodendrocyte development, they do not clearly define the functional differences between Olig1 and Olig2. We generated mice with a homozygous inactivation of Olig2. These mice did not feed and died on the day of birth. In the spinal cord of the mutant mice, motoneurons are largely eliminated and oligodendrocytes are not produced. Olig2(-/-) neuroepithelial cells in the ventral spinal cord failed to differentiate into motoneurons or oligodendrocytes and expressed an astrocyte marker, S100beta, at the time of oligodendrogenesis. Olig1 or Olig3, other family members, were expressed in the descendent cells that should have expressed Olig2. We concluded that Olig2 is an essential transcriptional regulator in motoneuron and oligodendrocyte development. Our data provide the first evidence that a single gene mutation leads to the loss of two cell types, motoneuron and oligodendrocyte.
Animals↗
Functional limitations of school-aged children seen in primary care.
OBJECTIVE: The purpose of the present study was to assess the prevalence of functional limitations in children seen in a large paediatric practice network and to identify sociodemographic, family and psychosocial factors related to functional limitations. STUDY DESIGN: Cross-sectional analysis. POPULATION: Children were recruited from two large, practice-based primary care research networks during their paediatric office visits. For the present study, participants included 14 630 school-aged children (ages 6-15 years) and their caregivers. OUTCOMES MEASURED: Parents completed written questionnaires including the Pediatric Symptom Checklist, the Family Apgar and the Functional Limitations Index. RESULTS: Findings indicated that 15% of children surveyed had some limitation in their daily functioning. More children had schoolwork and physical function limitations than limitations in personal and self-care. Logistic regression equations predicted functional limitations and health status in children from a model of sociodemographic factors, psychosocial symptoms and family functioning. CONCLUSIONS: A low but significant number of school-age children seen in the primary care setting experience functional limitations. Children with any psychosocial symptoms were at increased risk for functional limitations, indicating the critical need to screen for functional impairment in children with suspected behavioural or emotional problems. A screening tool of functional limitations may be useful for assessing the presence or absence of such limitations in children's daily function and warrants further investigation.
Activities of Daily Living↗
Interaction of selected vasodilating beta-blockers with adrenergic receptors in human cardiovascular tissues.
beta- And alpha 1-adrenoceptor antagonist properties of bufuralol, carvedilol, celiprolol, dilevalol, labetalol, and pindolol were investigated in human myocardium and mammary artery using binding techniques and functional studies. In myocardial membranes, beta-adrenoceptor antagonists showed monophasic competition isotherms for [125I]pindolol binding with high affinity (Ki from 1-100 nM), except for celiprolol which displayed a biphasic competition isotherm (pKi = 6.4 +/- 0.06 for beta 1- and 4.8 +/- 0.07 for beta 2-adrenoceptors). Drug interactions with alpha 1-adrenoceptors were evaluated in human mammary artery by [3H]prazosin binding and by measuring contractile responses to norepinephrine (NE). Labetalol and carvedilol showed a moderate affinity for alpha 1-adrenoceptors (pKi = 6.2 +/- 0.01 and 6.1 +/- 0.06, respectively), and inhibited NE-induced contractions (pA2 = 6.93 +/- 0.23 and 8.64 +/- 0.24, respectively). Dilevalol, bufuralol, and pindolol displayed weak effect both in binding (Ki in micromolar range) and functional experiments (pA2 = 5.98, 5.54, and 6.23, respectively). Celiprolol did not show antagonist properties up to 100 microM in functional studies, but displayed a slight affinity for alpha 1-adrenoceptors in binding studies. The data indicate that the vasodilating activity of these beta-adrenoceptor antagonists is caused in some instances by an alpha 1-adrenoceptor antagonism (labetalol, carvedilol), whereas for the others alternative mechanisms should be considered.
Adrenergic beta-Antagonists↗
Estimation of competitive antagonist affinity from functional inhibition curves using the Gaddum, Schild and Cheng-Prusoff equations.
1. The estimation of antagonist affinity from functional experiments in which the effect of a fixed agonist concentration is reduced by a range of antagonist concentrations ('functional inhibition curves') has been considered from both a theoretical and experimental viewpoint. 2. Theoretical predictions are compared with results obtained from the stimulation of [35S]-GTP gamma S binding by acetylcholine to membranes of Chinese hamster ovary (CHO) cells stably transfected with human m1-m4 muscarinic receptors, and inhibition of the stimulated binding by pirenzepine and AQ-RA 741. 3. The usual procedure of applying the Cheng-Prusoff correction is shown to be theoretically invalid, and predictions are made of the size and distribution of errors associated with this procedure. 4. A different procedure for estimating antagonist affinity, using the principles of dose-ratio analysis and analogous to use of the Gaddum equation, is found to be accurate and theoretically valid. 5. A novel method of analysis allows accurate estimation of both antagonist affinity and Schild slope, by fitting the combined data from an antagonist inhibition curve and an agonist activation curve directly to a form of the Schild equation (derived by Waud) using non-linear regression analysis. 6. It is shown that the conventional Schild analysis can be enhanced by treating part of the data as a family of inhibition curves and including in the Schild plot dose-ratios estimated from the inhibition curves.
Acetylcholine↗
Temporal integration in the presence of off-frequency maskers.
Temporal integration was measured at a relatively low and a relatively high signal frequency under conditions of off-frequency masking. The masker was typically gated for 300 ms, and the signal was presented 70 ms after masker onset. In experiment 1, the signal frequency was 500 or 2000 Hz. Temporal integration was measured in quiet and in the presence of a masker whose frequency was lower or higher than the signal frequency. In all listening situations, there was less integration at 2000 Hz than at 500 Hz. This effect of frequency was particularly dramatic in the presence of a lower frequency masker, where there was almost no integration at 2000 Hz. Experiment 2 showed that this dramatic effect of frequency cannot be understood in terms of the underlying psychometric functions. Experiment 3 measured temporal integration at 750 and 2000 Hz for a large number of masker-signal frequency separations for both a tonal and a noise masker, and in conditions where the masker was gated or continuous. The results with the gated tonal masker largely confirmed the results of experiment 1. The results with the continuous tonal masker and the gated or continuous noise masker, however, were quite different. In those cases, the amount of temporal integration at both signal frequencies was more or less independent of the masker-signal separation; the masked temporal integration was nearly equal to the integration in quiet. Thus based on the conditions evaluated here, off-frequency masked temporal integration differs substantially from integration in quiet only for gated tonal maskers located considerably lower in frequency than the signal. It is unclear how to account for this finding, although it may be related to attentional factors.
Adult↗
Use of invertebrate animals to teach physiological principles.
Experiments with invertebrate animals offer valuable opportunities in the teaching of physiology. In some cases, exercises that use these animals may demonstrate physiological principles more clearly than experiments that use vertebrates. Other experiments are easy to perform because of the latitude of conditions in which the tissues of many invertebrates function. Experiments with invertebrates can also illustrate a far wider range of physiological mechanisms than occurs in vertebrates and are especially suited for opportunities for independent original investigation by the students. Since at this time invertebrates are underutilized in teaching, much can be gained by the design, testing, and dissemination of innovative experimental protocols for student use.
Animal Welfare↗
Orthotopic lower urinary tract reconstruction in women using the Kock ileal neobladder: updated experience in 34 patients.
PURPOSE: Orthotopic lower urinary tract reconstruction has revolutionized urinary diversion following cystectomy. Initially performed solely in male patients, orthotopic diversion has now become a viable option in women. Currently, the orthotopic neobladder is the diversion of choice for women requiring lower urinary tract reconstruction at our institution. We evaluate and update our clinical and functional experience with orthotopic reconstruction in female patients. MATERIALS AND METHODS: Since June 1990, 34 women 31 to 86 years old (median age 67) have undergone orthotopic lower urinary tract reconstruction following cystectomy. Indications for cystectomy included transitional cell carcinoma in 29 patients, urachal adenocarcinoma in 1, mesenchymal tumor of endometrial origin in 1, cervical carcinoma in 1 and a fibrotic radiated bladder in 1. In addition, 1 woman underwent undiversion to the native urethra following a previous simple cystectomy and cutaneous diversion for eosinophilic cystitis. Data were analyzed according to postoperative early and late complications, survival, tumor recurrence, pathological evaluation of the cystectomy specimen, continence status, voiding pattern and patient satisfaction. The median followup in this group of patients was 30 months (range 17 to 70). RESULTS: There were no perioperative deaths, and 4 early (11%) and 3 (9%) late complications. Four patients died, none with a urethral recurrence, including 3 of metastatic bladder cancer and 1 of unrelated causes. In another patient with an extensive mesenchymal tumor of the uterus a sigmoid tumor recurred requiring conversion of the orthotopic reservoir to a cutaneous diversion. All of the remaining 29 patients are alive without evidence of disease. Intraoperative frozen section of the distal surgical margin (proximal urethra) accurately evaluated (confirmed by permanent section) the proximal urethra prospectively for tumor in all 29 specimens removed for transitional cell carcinoma, including 28 specimens (97%) without evidence of tumor and 1 specimen with carcinoma in situ. Complete daytime and nighttime continence was reported by 29 (88%) and 27 (82%) of 33 evaluable patients, respectively. A total of 28 patients (85%) void to completion, while 5 (15%) require some form of intermittent catheterization to empty the neobladder. Patient satisfaction is overwhelming. CONCLUSIONS: The excellent clinical and functional results demonstrated with further followup confirm our initial experience with orthotopic diversion in women. Careful selection of appropriate female candidates for orthotopic diversion is critical, and includes preoperative evaluation of the bladder neck and intraoperative frozen section analysis of the distal cystectomy margin. Furthermore, close monitoring of the retained urethra is mandatory in all women undergoing orthotopic diversion. We believe that the orthotopic neobladder is the urinary diversion of choice in women following cystectomy.
Adult↗
Modulations among the alerting, orienting and executive control networks.
This paper reports a series of experiments that were carried out in order to study the attentional system. Three networks make up this system, and each of them specializes in particular processes. The executive control network specializes in control processes, such as conflict resolution or detection of errors; the orienting network directs the processing system to the source of input and enhances its processing; the alerting network prepares the system for a fast response by maintaining an adequate level of activation in the cognitive system. Recently, Fan and collaborators [J Cogn Neurosci 14(3):340-347, 2002] designed a task to measure the efficiency of each network. We modified Fan's task to test the influences among the networks. We found that the executive control network is inhibited by the alerting network, whereas the orienting network raises the efficiency of the executive control network (Experiment 1). We also found that the alerting network influences the orienting network by speeding up its time course function (Experiment 2). Results were replicated in a third experiment, proving the effects to be stable over time, participants and experimental context, and to be potentially important as a tool for neuropsychological assessment.
Acoustic Stimulation↗
Modulo is a target of Myc selectively required for growth of proliferative cells in Drosophila.
In Drosophila, the homologue of the proto-oncogene Myc is a key regulator of both cell size and cell growth. The identities and roles of dMyc target genes in these processes, however, remain largely unexplored. Here, we investigate the function of the modulo (mod) gene, which encodes a nucleolus localized protein. In gain of function or loss of function experiments, we demonstrate that mod is directly controlled by dMyc. Strikingly, in proliferative imaginal cells, mod loss-of-function impairs both cell growth and cell size, whereas larval endoreplicative tissues grow normally. In contrast to dMyc, over-expressing Mod in wing imaginal discs is not sufficient to induce cell growth. Taken together, our results indicate that mod does not possess the full spectrum of dMyc activities, but is required selectively in proliferative cells to sustain their growth and to maintain their specific size.
Animals↗
Hox11 controls the genesis of the spleen.
Many homeobox genes are clustered in a linear array along a chromosome, reflecting their ordered expression along the anterior-posterior axis of the embryo. Expression patterns as well as grafting, ectopic expression and loss-of-function experiments suggest that the Hox genes encode a combinatorial system of positional specification along that axis. In contrast, the function of orphan homeobox genes located at sites outside the four mammalian Hox clusters is less well understood. To assess the functional role of the orphan homeobox gene Hox11, we have generated Hox11-deficient mice through gene targeting. Hox11-/- mice have no spleen, but otherwise appear normal. Hox11 is normally expressed in the splenic anlage arising from the splanchnic mesoderm. Hox11-/- embryos have no cellular organization at the site of splenic development but all other splanchnic derivatives develop normally. Hox11 controls the genesis of a single organ, providing new insight into the genetic regulation of morphogenesis.
Animals↗
Structure of the activation domain of the GM-CSF/IL-3/IL-5 receptor common beta-chain bound to an antagonist.
Heterodimeric cytokine receptors generally consist of a major cytokine-binding subunit and a signaling subunit. The latter can transduce signals by more than 1 cytokine, as exemplified by the granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin-2 (IL-2), and IL-6 receptor systems. However, often the signaling subunits in isolation are unable to bind cytokines, a fact that has made it more difficult to obtain structural definition of their ligand-binding sites. This report details the crystal structure of the ligand-binding domain of the GM-CSF/IL-3/IL-5 receptor beta-chain (beta(c)) signaling subunit in complex with the Fab fragment of the antagonistic monoclonal antibody, BION-1. This is the first single antagonist of all 3 known eosinophil-producing cytokines, and it is therefore capable of regulating eosinophil-related diseases such as asthma. The structure reveals a fibronectin type III domain, and the antagonist-binding site involves major contributions from the loop between the B and C strands and overlaps the cytokine-binding site. Furthermore, tyrosine(421) (Tyr(421)), a key residue involved in receptor activation, lies in the neighboring loop between the F and G strands, although it is not immediately adjacent to the cytokine-binding residues in the B-C loop. Interestingly, functional experiments using receptors mutated across these loops demonstrate that they are cooperatively involved in full receptor activation. The experiments, however, reveal subtle differences between the B-C loop and Tyr(421), which is suggestive of distinct functional roles. The elucidation of the structure of the ligand-binding domain of beta(c) also suggests how different cytokines recognize a single receptor subunit, which may have implications for homologous receptor systems. (Blood. 2000;95:2491-2498)
Antibodies, Monoclonal↗
Macrophage prostaglandin production contributes to the age-associated decrease in T cell function which is reversed by the dietary antioxidant vitamin E.
The aging process is associated with a decline in T cell-mediated immunity, including decreased interleukin (IL)-2 production and mitogen-induced T cell proliferation. Because macrophages (M phi) from old mice have higher production of prostaglandin (PG) E2 than young mice, and PGE2 has been shown to suppress T cell-mediated function, we hypothesized that increased production of PGE2 would contribute to decreased T cell function with aging and that decrease in PGE2 production by dietary antioxidants would enhance T cell-mediated function. Experiments were conducted in which combinations of purified M phi and T cells (> 95% pure) from young or old C57BL/6N1A mice were cultured together. Co-cultures containing T cells and M phi from old mice had reduced ConA-stimulated proliferation and IL-2 secretion than those consisting of T cells and M phi from young mice. Addition of M phi from old mice suppressed proliferation and IL-2 secretion by T cells from young mice. Likewise, T cells from old mice secreted more IL-2 when cultured with M phi from young mice compared to those cultured with M phi from old mice. Addition of PGE2, at concentrations produced by old M phi, decreased proliferation and IL-2 production by young but not old T cells. Neither addition of H2O2 at physiological levels, nor catalase changed the response of cultures from young or old mice. However, addition of indomethacin and the antioxidant nutrient vitamin E, both of which decreased PGE2 production, improved T cell proliferation and IL-2 production. These experiments demonstrate that increased production of PGE2 by M phi contributes to the age-associated decline in T cell function. Vitamin E improves T cell responsiveness in old mice mostly by reducing M phi PGE2 production, although a direct effect of vitamin E on T cells was also observed.
Aging↗
The Arabidopsis TT2 gene encodes an R2R3 MYB domain protein that acts as a key determinant for proanthocyanidin accumulation in developing seed.
In Arabidopsis, proanthocyanidins specifically accumulate in the endothelium during early seed development. At least three TRANSPARENT TESTA (TT) genes, TT2, TT8, and TTG1, are necessary for the normal expression of several flavonoid structural genes in immature seed, such as DIHYDROFLAVONOL-4-REDUCTASE and BANYULS (BAN). TT8 and TTG1 were characterized recently and found to code for a basic helix-loop-helix domain transcription factor and a WD-repeat-containing protein, respectively. Here the molecular cloning of the TT2 gene was achieved by T-DNA tagging. TT2 encoded an R2R3 MYB domain protein with high similarity to the rice OsMYB3 protein and the maize COLORLESS1 factor. A TT2-green fluorescent protein fusion protein was located mostly in the nucleus, in agreement with the regulatory function of the native TT2 protein. TT2 expression was restricted to the seed during early embryogenesis, consistent with BAN expression and the proanthocyanidin deposition profile. Finally, in gain-of-function experiments, TT2 was able to induce ectopic expression of BAN in young seedlings and roots in the presence of a functional TT8 protein. Therefore, our results strongly suggest that stringent spatial and temporal BAN expression, and thus proanthocyanidin accumulation, are determined at least partially by TT2.
Alleles↗