Steroid hormones, energetic state, and immunocompetence vary across reproductive contexts in a parthenogenetic lizard.

Reproduction is energetically expensive and investing in this life history trait is likely accompanied by significant changes in physiological activity. Investment strategy necessary for achieving reproductive success in reptiles can vary with reproductive form and pattern, potentiating different consequences for competing fitness-related traits such as those key to survival. The goal of this study was to assess if and how energetic state (i.e., energy metabolites) and self-maintenance (i.e., immunocompetence) are hormonally modulated across reproductive contexts in an oviparous, parthenogenetic lizard, the Colorado Checkered Whiptail Aspidoscelis neotesselata. Here blood plasma samples were collected from lizards within the US Army Fort Carson Military Installation near Colorado Springs, CO, USA, during seasons of reproductive activity (i.e., June) and inactivity (i.e., August). Measures of reproductive (i.e., estradiol) and energy-mobilizing (i.e., corticosterone) hormones, energy metabolites (i.e., glucose, triglycerides, and free glycerol), and innate immunity (i.e., bactericidal ability) were compared by season and reproductive stage. Levels of energy metabolites and bactericidal ability were compared to levels of E2 and CORT. Bactericidal ability was also compared to levels of energy metabolites. Corticosterone and glucose levels were lower during the reproductive season while triglyceride levels and bactericidal ability were higher, but both estradiol and free glycerol levels did not differ between seasons. Throughout vitellogenesis, corticosterone and glucose levels as well as bactericidal ability did not differ, but estradiol levels were higher during early and mid-stage and both triglyceride and free glycerol levels were lower during gravidity. Corticosterone levels were negatively associated with circulating triglycerides and bactericidal ability, but were not related to glucose nor free glycerol levels. Estradiol levels were positively associated with free glycerol levels and bactericidal ability, but were not related to glucose nor triglyceride levels. Finally, bactericidal ability was negatively associated with glucose, but positively associated with triglycerides. Differences in energetic state and immunocompetence are thus reflected by shifts in hormone secretion across reproductive investment. These findings provide partial support for the hypothesis that energetic state is differentially regulated by steroid hormones to afford reproduction, potentially at the cost of future survival.

Transcranial Doppler Ultrasound Detection of Microemboli as a Predictor of Cerebral Events in Patients with Symptomatic and Asymptomatic Carotid Disease: A Systematic Review and Meta-Analysis.

OBJECTIVE: Identification of patients who will benefit from carotid endarterectomy is not entirely effective, primarily utilising degree of carotid stenosis. This study aimed at determining if microembolic signals (MES) detected by transcranial Doppler ultrasound (TCD) can provide clinically useful information regarding stroke risk in patients with carotid atherosclerosis. METHODS: A meta-analysis of prospective studies was performed. Three analyses were proposed investigating MES detection as a predictor of: stroke or TIA, stroke alone, and stroke or TIA but with an increased positivity threshold. Subgroup analysis was used to compare pre-operative (symptomatic or asymptomatic) patients and peri- or post-operative patients. RESULTS: Twenty-eight studies reported data regarding both MES status and neurological outcome. Of these, 22 papers reported data on stroke and TIA as an outcome, 19 on stroke alone, and eight on stroke and TIA with increased positivity threshold. At the median pre-test probability of 3.0%, the post-test probabilities of a stroke after a positive and negative TCD were 7.1% (95% CI 5-10.1) and 1.2% (95% CI 0.6-2.5), respectively. In addition, the sensitivities and specificities of each outcome showed that increasing the threshold for positivity to 10 MES per hour would make TCD a more clinically useful tool in peri- and post-operative patients. CONCLUSION: TCD provides clinically useful information about stroke risk for patients with carotid disease and is technically feasible in most patients. However, the generally weak level of evidence constituting this review means definitive recommendations cannot be made.

Post-synaptic GABA(B) receptors--possible controllers of coincidence detection?

Post-synaptic GABA(B) responses (slow, late hyperpolarisations which can be eliminated by perfusion with phaclofen) can be recorded in vitro from many, but not all, neurones in the intermediate medial hyperstriatum ventrale (IMHV). The IMHV is an area of the chick forebrain which is remarkable for its plasticity, and for its essential role in two specific types of early learning-imprinting, and a form of one-trial passive-avoidance learning. Post-synaptic GABA(B) responses are strongly statistically associated with other properties (such as high membrane resistance) which are, themselves, dependent on a bird's past history. There is also evidence that their incidence changes with prior training in vivo and with age. GABA(B) hyperpolarisations are always offset to a varying extent by excitatory NMDA components. These two components follow a very similar time-course, so that the duration and (to a lesser extent), the magnitude of a response is controlled by the balance between the two systems. The evidence suggests that this balance fluctuates, and that its fluctuations determine the extent to which any neurone can function as a coincidence detector.

The diagnosis and consequences of Stickler syndrome.

The objective was to study the expressivity of Stickler syndrome in affected children and adults in the UK and to highlight issues for improving early diagnosis, treatment and counselling. A postal questionnaire survey of the 216 members of the Stickler Syndrome Support Group was carried out. Of the 153 (71%) who responded to the questionnaire, 48 (61%) of adults and 15 (20%) of children had experienced retinal detachment; 36 (49%) of the children and 18 (23%) of the adults were born with a cleft palate. Only 5 (7%) of the children and none of the adults had been diagnosed by a cleft surgeon, although 23 (31%) of the children had been diagnosed originally as having Pierre-Robin sequence. Only a third of the adults had been given any genetic counselling. Stickler syndrome is an under-diagnosed condition with profound consequences, particularly with respect to vision. Earlier diagnosis by the cleft team may help to reduce suffering and increase awareness of the condition.

Low-threshold N-methyl-D-aspartate receptor function correlates negatively with learning.

The intermediate, medial hyperstriatum ventrale (IMHV) is an area of the forebrain of the domestic chick which exhibits great plasticity. Moreover, there is a strong link between plasticity in the IMHV and specific changes in behaviour. The IMHV in vitro is still plastic, and many of its physiological properties are age-dependent, peaking in slices taken from 3- or 4-day-old birds. This 'window' coincides with an important transitional period in a chick's normal behavioural development. It has also been claimed that reversal training is at its most effective in 3- and 4-day-old birds - a proposition which was confirmed by the experiments reported here. A combination of in vivo training followed by in vitro electrophysiology also revealed that the function of low-threshold N-methyl-D-aspartate receptors (one of the age-related variables) is negatively related to the effectiveness of reversal training, when age is held constant.

Effects of nitric oxide release in an area of the chick forebrain which is essential for early learning.

Extracellular recording techniques were used to study the effects of the nitric oxide releasing agents diethylamine-NO (DEA-NO) and S-nitroso-N-acetyl-penicillamine (SNAP) on synaptic transmission in the intermediate and medial part of the hyperstriatum ventrale (IMHV), a part of the domestic chick forebrain that is essential for some forms of early learning. The field response evoked by local electrical stimulation was recorded in the IMHV in an in vitro slice preparation. DEA-NO (100-200 mgr) significantly depressed the field response in a concentration dependent and reversible manner. However, the depression produced by perfusion with 400 mgr DEA-NO, was not reversed following washout of the drug. With 400 mgr DEA-NO, NO reaches a maximum concentration of 10 mgr at 2 min of perfusion, and then declines slowly. SNAP (400 mgr) produced an effect similar to 400 mgr DEA-NO. Neither the immediate nor the longer-term depressive effect of NO is mediated by activation of guanylyl cyclase because in the presence of both low and high doses of ODQ, a potent and selective inhibitor of NO-stimulated guanylyl cyclase, NO produced the same depression of the field response. There is evidence however that the IMHV possesses c-GMP responsive elements since direct perfusion of 8-Br-cGMP (1 mM) produced a long-term but not an immediate depression. The long-term depression produced by 400 mgr DEA-NO was eliminated in the presence of either a selective adenosine A(1) receptor antagonist or an ADP-ribosyltransferase inhibitor. It was also possible to prevent the long-term effect in the presence of tetraethyl ammonium a K(+)-channel blocker. These results suggest that the NO may be acting presynaptically in a synergistic fashion with the adenosine A(1) receptor to depress transmitter release.

Cytomegalovirus IE2 protein stimulates interleukin 1beta gene transcription via tethering to Spi-1/PU.1.

Potent induction of the gene coding for human prointerleukin 1beta (il1b) normally requires a far-upstream inducible enhancer in addition to a minimal promoter located between positions -131 and +12. The transcription factor Spi-1 (also called PU.1) is necessary for expression and binds to the minimal promoter, thus providing an essential transcription activation domain (TAD). In contrast, infection by human cytomegalovirus (HCMV) can strongly activate il1b via the expression of immediate early (IE) viral proteins and eliminates the requirement for the upstream enhancer. Spi-1 has been circumstantially implicated as a host factor in this process. We report here the molecular basis for the direct involvement of Spi-1 in HCMV activation of il1b. Transfection of Spi-1-deficient HeLa cells demonstrated both the requirement of Spi-1 for IE activity and the need for a shorter promoter (-59 to +12) than that required in the absence of IE proteins. Furthermore, in contrast to normal, enhancer-dependent il1b expression, which absolutely requires both the Spi-1 winged helix-turn-helix (wHTH) DNA-binding domain and the majority of the Spi-1 TAD, il1b expression in the presence of IE proteins does not require the Spi-1 TAD, which plays a synergistic role. In addition, we demonstrate that a single IE protein, IE2, is critical for the induction of il1b. Protein-protein interaction experiments revealed that the wing motif within the Spi-1 wHTH domain directly recruits IE2. In turn, IE2 physically associates with the Spi-1 wing and requires the integrity of at least one region of IE2. Functional analysis demonstrates that both this region and a carboxy-terminal acidic TAD are required for IE2 function. Therefore, we propose a protein-tethered transactivation mechanism in which the il1b promoter-bound Spi-1 wHTH tethers IE2, which provides a TAD, resulting in the transactivation of il1b.

Identification of a competitive translation determinant in the 3' untranslated region of alfalfa mosaic virus coat protein mRNA.

We report that the competitive translational activity of alfalfa mosaic virus coat protein mRNA (CP RNA), a nonadenylated mRNA, is determined in part by the 3' untranslated region (UTR). Competitive translation was characterized both in vitro, with cotranslation assays, and in vivo, with microinjected Xenopus laevis oocytes. In wheat germ extracts, coat protein synthesis was constant when a fixed amount of full-length CP RNA was cotranslated with increasing concentrations of competitor globin mRNA. However, translation of CP RNA lacking the 3' UTR decreased significantly under competitive conditions. RNA stabilities were equivalent. In X. laevis oocytes, which are translationally saturated and are an inherently competitive translational environment, full-length CP RNA assembled into large polysomes and coat protein synthesis was readily detectable. Alternatively, CP RNA lacking the 3' UTR sedimented as small polysomes, and little coat protein was detected. Again, RNA stabilities were equivalent. Site-directed mutagenesis was used to localize RNA sequences or structures required for competitive translation. Since the CP RNA 3' UTR has an unusually large number of AUG nucleotide triplets, two AUG-containing sites were altered in full-length RNA prior to oocyte injections. Nucleotide substitutions at the sequence GAUG, 20 nucleotides downstream of the coat protein termination codon, specifically reduced full-length CP RNA translation, while similar substitutions at the next AUG triplet had little effect on translation. The competitive influence of the 3' UTR could be explained by RNA-protein interactions that affect translation initiation or by ribosome reinitiation at downstream AUG codons, which would increase the number of ribosomes committed to coat protein synthesis.

Morphological and electrophysiological properties of neurons in an area of the chick brain involved in learning.

The intermediate, medial part of the hyperstriatum ventrale (IMHV) is an area of the avian forebrain which is essential for two forms of early learning in the domestic chick. We have developed an in vitro slice preparation which contains part of the IMHV and have found that the electrophysiological properties of the area show a considerable degree of plasticity. In particular, age and prior learning appear to modify the properties of single neurons recorded intracellularly. We have used the in vitro slice preparation to make intracellular recordings from 38 single neurons in the IMHV and have then dye-injected each cell to find out whether there is any relationship between electrophysiological and morphological characteristics. The basic membrane properties of each neuron were measured. Responses to standard electrical stimuli, delivered extracellularly, were also recorded, and each neuron was classified on this basis. Finally, the presence or absence of spontaneously occurring bursts of EPSPs was noted. At the end of recording biocytin was injected into the cell. After the tissue had been processed, each cell was drawn. The area of the cell body was measured, the number of dendrites was counted, and dendritic extent and branching were estimated. Each cell was also classified as 'spiny' or 'non-spiny'. We found that neurons displaying one particular type of response to external stimulation possessed a well defined set of morphological and electrical properties. In addition, three parameters--electrical resistance, somatic area, and the presence or absence of dendritic spines--were related to specific subsets of anatomical and physiological characteristics. The possible relevance of these findings to the plasticity of the IMHV is discussed.

A novel STAT-like factor mediates lipopolysaccharide, interleukin 1 (IL-1), and IL-6 signaling and recognizes a gamma interferon activation site-like element in the IL1B gene.

Binding of many cytokines to their cognate receptors immediately activates Jak tyrosine kinases and their substrates, STAT (signal transducers and activators of transcription) DNA-binding proteins. The DNA binding targets of STATs are sequence elements related to the archetypal gamma interferon activation site, GAS. However, association of interleukin 1 (IL-1) with Jak-STAT signaling has remained unresolved. We now report an element termed LILRE (lipopolysaccharide [LPS] and IL-1-responsive element) in the human prointerleukin 1beta gene (IL1B) which can be immediately induced by either lipopolysaccharide (LPS) or IL-1 protein to bind a tyrosine-phosphorylated protein. This LPS- and IL-1-induced factor (LIL factor) is recognized by an antibody raised against the N terminus of Stat1, but not by those specific for either the C terminus of Stat1 or any other GAS-binding STAT. Phosphotyrosine (P-Tyr) specifically inhibits formation of the LIL factor-DNA complex, suggesting the importance of P-Tyr for the DNA-binding activity, as has been found for all STAT dimers. Analysis of DNA-binding specificity demonstrates that the LIL factor possesses a novel GAS-like binding activity that contrasts with those of other STATs in a requirement for a G residue at position 8 (TTCCTGAGA). Further investigation has revealed that IL-6, but neither IL-4 nor gamma interferon, activates the LIL factor. Thus, the existence of such a STAT-like factor (LIL-Stat) relates the LPS and IL-1 signaling pathway to other cytokine receptor signaling pathways via the activation of STATs. Moreover, the unique DNA-binding specificity and antigenicity of this factor suggest that LPS, IL-1, and IL-6 may use a common signaling pathway.

Electrophysiological correlates of prior training: an in vitro study of an area of the avian brain which is essential for early learning.

Day-old domestic chicks will peck at any small, distinct object, such as a metal bead. One-trial passive avoidance learning can be established by coating the metal bead with methyl anthranilate (MeA) and allowing the birds to peck it once, after which they conspicuously avoid it. We have used birds successfully trained not to peck metal beads, and a control set of chicks where the training beads were innocuously dipped in water. Brain slices were prepared from both groups, containing the left, intermediate, medial part of the hyperstriatum ventrale (IMHV)--a region essential for this form of early learning. The electrophysiological properties of neurones in the IMHV were examined in vitro. Neurones recorded intracellularly in slices taken from MeA-trained birds had higher membrane resistances than did cells from water-trained controls. MeA training was also associated with an increased incidence of spontaneous, large EPSPs. Field responses to local electrical stimulation appeared to be somewhat greater in MeA-trained birds than in water-trained controls. In contrast, field potentials proved harder to potentiate with a burst of relatively high frequency stimulation in MeA-trained birds: the change in amplitude was less in MeA-trained birds, and there was less variability than in slices from water-trained controls.

Age and the effects of 2-D,L-amino-5-phosphonovalerate in an area of the chick forebrain which is essential for early learning.

The intermediate, medial part of the hyperstriatum ventrale (IMHV) is a region of the avian forebrain which is known to be essential for early learning in the domestic chick. The IMHV in an in vitro slice preparation displays two forms of synaptic plasticity. The incidence of both varies with age and is maximal between 3 and 5 days post-hatch. Since NMDA receptors are critical for at least one of these plasticities, we have investigated the relationship between age and the contribution of NMDA receptors to the field response evoked by local, low-frequency stimulation and have found that the magnitude of the NMDA-dependent component of the response varies with age peaking between 3 and 5 days post-hatch. Spontaneous neural activity, recorded intracellularly, can be completely and reversibly silenced by NMDA receptor blockade and the incidence of spontaneous activity also varies with age, peaking between 3 and 5 days. These results suggest that the IMHV contains NMDA receptors which can be activated near resting membrane potential. Either the efficiency or the numbers of these receptors is maximal at a specific point in development and their peak activity coincides with a peak in synaptic plasticity. These characteristics are similar to those reported for young mammals.

Noradrenaline and potentiation in the chick brain slice.

The role of the noradrenergic system as a modulator of neurotransmission in the chick forebrain was investigated in brain slices containing the medial hyperstriatum ventrale, an area known to be involved in learning and memory. The alpha 2-agonist clonidine (20 microM) decreased the post-synaptic response to local stimulation at 0.1 Hz, while activation of beta 2 receptors increased this response. Induction of persistent potentiation following stimulation at 5 Hz was blocked by drugs (20 microM ICI 118,551; 20 microM propranolol) which showed preferential antagonistic activity at the beta 2 receptor but not by the beta 1-preferential antagonist timolol. This effect may be due to an interaction with the NMDA receptor system.

Monocyte expression of the human prointerleukin 1 beta gene (IL1B) is dependent on promoter sequences which bind the hematopoietic transcription factor Spi-1/PU.1.

Interleukin-1 beta (IL-1 beta) is produced primarily by stimulated monocytes, suggesting that the IL1B gene, which codes for this protein, depends upon at least one cell-type-specific factor. Our previous characterization of the IL1B promoter indicated that the region between -131 and +12 is sufficient to direct cell-type-specific expression of a reporter gene (F. Shirakawa, K. Saito, C.A. Bonagura, D.L. Galson, M.J. Fenton, A.C. Webb, and P. E. Auron, Mol. Cell. Biol. 13:1332-1344, 1993). We now show that a sequence located between positions -50 and -39 of the IL1B promoter binds the tissue-restricted Ets domain transcription factor Spi-1/PU.1 (Spi-1). Mutation of this site abrogates binding of this factor and reduces the ability of the IL1B promoter to function in macrophages. A second Spi-1 binding site located between positions -115 and -97 also is required for maximal IL1B promoter activity in the presence of the proximal Spi-1 binding site. In addition, an activation domain-deficient Spi-1 expression vector acts as a dominant-negative inhibitor of reporter gene expression in a monocyte cell line. Finally, the IL1B promoter, which is inactive in Spi-1-deficient HeLa cells, is activated in these cells by cotransfection with a Spi-1 expression vector. Thus, the cell-type-specific expression of the IL1B promoter appears to be dependent on the binding of Spi-1.

Transcription factors NF-IL6 and CREB recognize a common essential site in the human prointerleukin 1 beta gene.

A site located between -2782 and -2729 of the human prointerleukin-1 beta (IL1B) gene functions as a strong lipopolysaccharide (LPS)-responsive enhancer independent of the previously identified enhancer located between -2896 and -2846 (F. Shirakawa, K. Saito, C.A. Bonagura, D.L. Galson, M. J. Fenton, A. C. Webb, and P. E. Auron, Mol. Cell. Biol. 13:1332-1344, 1993). Although these two enhancers appear to function cooperatively in the native sequence context, they function independently as LPS-responsive elements upon removal of an interposed silencer sequence. The new enhancer is not induced by dibutyryl cyclic AMP (dbcAMP) alone but is superinduced by costimulation with LPS-dbcAMP. This pattern of induction depends upon the nature of the sequence, a composite NF-IL6-cAMP response element (CRE) binding site. This pseudosymmetrical sequence is shown to contrast with a classical symmetric CRE which responds to dbcAMP but not LPS. DNA binding studies using in vivo nuclear extract, recombinant proteins, and specific antibodies show that LPS induces the formation of two different complexes at the enhancer: (i) an NF-IL6-CREB heterodimer and (ii) a heterodimer consisting of NF-IL6 and a non-CREB, CRE-binding protein. Cotransfection studies using NF-IL6 and CREB expression vectors show that NF-IL6 transactivates the enhancer in the presence of LPS, whereas CREB acts either positively or negatively, depending upon its cAMP-regulated phosphorylation state. Our data demonstrate that the newly identified enhancer is a specialized LPS-responsive sequence which can be modulated by cAMP as a result of the involvement of NF-IL6-CRE-binding protein heterodimers.

Effects of light hatching on synapse number and size in the intermediate and medial part of the hyperstriatum ventrale of the domestic chick.

The effects of pre-hatch light exposure on synaptic development in the intermediate and medial part of the hyperstriatum ventrale (IMHV) of the chick brain were investigated. Quantitative electron microscopical techniques were used to determine the size and numerical density of synapses and it was seen that in light hatched chicks there was a significant increase in the density of synapses in the left IMHV but that the size of synapses in these birds was decreased. These results provide a link between synaptic development and plasticity.

Human cathepsin S: chromosomal localization, gene structure, and tissue distribution.

The human lysosomal cysteine proteinases, cathepsins H, L, and B, have been mapped to chromosomes 15, 9, and 8, respectively, and the genomic structures of cathepsins L and B have been determined. We report here the chromosomal localization and partial gene structure for a recently sequenced human cysteine proteinase, cathepsin S. A 20-kilobase pair genomic clone of the human cathepsin S gene was isolated from a human fibroblast genomic library and used to map the human cathepsin S gene to chromosome 1q21 by fluorescence in situ hybridization. This clone contains exons 1 through 5, introns 1 through 4, part of intron 5, and > 7 kilobase pairs of the 5'-flanking sequence. The gene structure of human cathepsin S is similar to that of cathepsin L through the first 5 exons, except that cathepsin S introns are substantially larger. Sequencing of the 5'-flanking region revealed, similar to human cathepsin B, no classical TATA or CAAT box. In contrast to cathepsin B, cathepsin S contains only two SP1 and at least 18 AP1 binding sites that potentially could be involved in regulation of the gene. This 5'-flanking region also contains CA microsatellites. The presence of AP1 sites and CA microsatellites suggest that cathepsin S can be specifically regulated. Results of Northern blotting using probes for human cathepsins B, L, and S are consistent with this hypothesis; only cathepsin S shows a restricted tissue distribution, with highest levels in spleen, heart, and lung. In addition, immunostaining of lung tissue demonstrated detectable cathepsin S only in lung macrophages. The high level of expression in the spleen and in phagocytes suggests that cathepsin S may have a specific function in immunity, perhaps related to antigen processing.

NMDA-receptors and potentiation in an area of avian brain essential for learning.

The intermediate medial part of the hyperstriatum ventrale (IMHV), part of the avian forebrain, is essential for early learning in the domestic chick. Persistent potentiation (PP) can sometimes be induced in the IMHV in vitro. One can predict success from events which occur during the stimulation (1 min at 5 Hz) which is used for induction: the original response must be transiently replaced by a later, slower response (the LPSR). The LPSR has a comparatively high threshold of activation, its rate of development is inversely related to magnesium concentration and it can be eliminated by NMDA antagonists, as can the induction of PP. PP in the IMHV is therefore dependent upon NMDA receptors and the LPSR represents the activation of these receptors.