Frequency-domain hot-wire sensor and 3D model for thermal conductivity measurements of reactive and corrosive materials at high temperatures.

High temperature solids and liquids are becoming increasingly important in next-generation energy and manufacturing systems that seek higher efficiencies and lower emissions. Accurate measurements of thermal conductivity at high temperatures are required for the modeling and design of these systems, but commonly employed time-domain measurements can have errors from convection, corrosion, and ambient temperature fluctuations. Here, we describe the development of a frequency-domain hot-wire technique capable of accurately measuring the thermal conductivity of solid and molten compounds from room temperature up to 800 °C. By operating in the frequency-domain, we can lock into the harmonic thermal response of the material and reject the influence of ambient temperature fluctuations, and we can keep the probed volume below 1 µl to minimize convection. The design of the microfabricated hot-wire sensor, electrical systems, and insulating wire coating to protect against corrosion is covered in detail. Furthermore, we discuss the development of a full three-dimensional multilayer thermal model that accounts for both radial conduction into the sample and axial conduction along the wire and the effect of wire coatings. The 3D, multilayer model facilitates the measurement of small sample volumes important for material development. A sensitivity analysis and an error propagation calculation of the frequency-domain thermal model are performed to demonstrate what factors are most important for thermal conductivity measurements. Finally, we show thermal conductivity measurements including model data fitting on gas (argon), solid (sulfur), and molten substances over a range of temperatures.

Body mass index, waist-to-hip ratio, waist circumference and waist-to-height ratio cannot predict male semen quality: a report of 1231 subfertile Chinese men.

There were controversial results between obesity-associated markers and semen quality. In this study, we investigated the correlations between age, obesity-associated markers including body mass index (BMI), waist-to-hip ratio (WHR), waist-to-height ratio (WHtR) and waist circumference (WC), the combination of age and obesity-associated markers, semen parameters and serum reproductive hormone levels in 1231 subfertile men. The results showed that BMI, WC, WHR and WHtR were positively related to age, and there were also positive relations between BMI, WHR, WC and WHtR and between sperm concentration (SC), total sperm count (TSC), progressive motility (PR), sperm motility and per cent of normal sperm morphology (NSM). However, age, each of obesity-associated markers and the combination of obesity-associated markers and age were unrelated to any of semen parameters including total normal-progressively motile sperm count (TNPMS). Age, BMI, WHR, WC and WHtR were negatively related to serum testosterone and SHBG levels. However, only serum LH and FSH levels were negatively related to sperm concentration, NSM and sperm motility. In a conclusion, although age and obesity have significant impacts on reproductive hormones such as testosterone, SHBG and oestradiol, semen parameters related to FSH and LH could not be influenced, indicating that obesity-associated markers could not predict male semen quality.

Stage and cell-specific expression of calmodulin-dependent phosphodiesterases in mouse testis.

Calcium and cyclic nucleotides are second messengers that regulate the development and functional activity of spermatozoa. Calcium/calmodulin-dependent phosphodiesterases (CaM-PDEs) are abundant in testicular cells and in mature spermatozoa and provide one means by which calcium regulates cellular cyclic nucleotide content. We examined the spatial and temporal expression profiles of three knownCaM-PDE genes, PDE1A, PDE1B, and PDE1C, in the testis. In situ hybridization and immunofluorescent staining showed that both PDE1A and PDE1C are highly expressed but at different stages in developing germ cells. However, a very low hybridization signal of PDE1B exists uniformly throughout the seminiferous epithelium and the interstitium. More specifically, PDE1A mRNA is found in round to elongated spermatids, with protein expression in the tails of elongated and maturing spermatids. In contrast, PDE1C mRNA accumulates during early meiotic prophase and throughout meiotic and postmeiotic stages. Immunocytochemistry showed a diffuse, presumably cytosolic distribution of the expressed protein. The distinct spatial and temporal expression patterns of CaM-PDEs suggest important but different physiological roles for these CaM-PDEs in developing and mature spermatozoa.

Leptin induces insulin-like signaling that antagonizes cAMP elevation by glucagon in hepatocytes.

Although many effects of leptin are mediated through the central nervous system, leptin can regulate metabolism through a direct action on peripheral tissues, such as fat and liver. We show here that leptin, at physiological concentrations, acts through an intracellular signaling pathway similar to that activated by insulin in isolated primary rat hepatocytes. This pathway involves stimulation of phosphatidylinositol 3-kinase (PI3K) binding to insulin receptor substrate-1 and insulin receptor substrate-2, activation of PI3K and protein kinase B (AKT), and PI3K-dependent activation of cyclic nucleotide phosphodiesterase 3B, a cAMP-degrading enzyme. One important function of this signaling pathway is to reduce levels of cAMP, because leptin-mediated activation of both protein kinase B and phosphodiesterase 3B is most marked following elevation of cAMP by glucagon, and because leptin suppresses glucagon-induced cAMP elevation in a PI3K-dependent manner. There is little or no expression of the long form leptin receptor in primary rat hepatocytes, and these signaling events are probably mediated through the short forms of the leptin receptor. Thus, leptin, like insulin, induces an intracellular signaling pathway in hepatocytes that culminates in cAMP degradation and an antagonism of the actions of glucagon.

Sparing effect of leptin on liver glycogen stores in rats during the fed-to-fasted transition.

The effect of moderate hyperleptinemia ( approximately 20 ng/ml) on liver and skeletal muscle glycogen metabolism was examined in Wistar rats. Animals were studied approximately 90 h after receiving recombinant adenoviruses encoding rat leptin (AdCMV-leptin) or beta-galactosidase (AdCMV-betaGal). Liver and skeletal muscle glycogen levels in the fed and fasted (18 h) states were similar in AdCMV-leptin- and AdCMV-betaGal-treated rats. However, after delivery of a glucose bolus, liver glycogen levels were significantly greater in AdCMV-leptin compared with AdCMV-betaGal rats (P < 0.05). To investigate the mechanism(s) of these differences, glycogen levels were measured immediately after the cessation of a 3- or 6-h glucose infusion or 3, 6, and 9 h after the cessation of a 6-h glucose infusion. Similar increases in liver and skeletal muscle glycogen occurred in hyperleptinemic and control rats in response to glucose infusions. However, 3 and 6 h after the cessation of a glucose infusion, liver glycogen levels were approximately twofold greater (P < 0.05) in AdCMV-leptin-treated compared with AdCMV-betaGal-treated animals. Skeletal muscle glycogen levels were similar in AdCMV-leptin-treated and AdCMV-betaGal-treated animals at the same time points. Glycogen phosphorylase, phosphodiesterase 3B, and glycogen synthase activities were unaltered by hyperleptinemia. We conclude that moderate increases in plasma leptin levels decrease liver glycogen degradation during the fed-to-fasted transition.

Phosphorylation and inhibition of olfactory adenylyl cyclase by CaM kinase II in Neurons: a mechanism for attenuation of olfactory signals.

Acute desensitization of olfactory signaling is a critical property of the olfactory system that allows animals to detect and respond to odorants. Correspondingly, an important feature of odorant-stimulated cAMP increases is their transient nature, a phenomenon that may be attributable to the unique regulatory properties of the olfactory adenylyl cyclase (AC3). AC3 is stimulated by receptor activation and inhibited by Ca2+ through Ca2+/calmodulin kinase II (CaMKII) phosphorylation at Ser-1076. Since odorant-stimulated cAMP increases are accompanied by elevated intracellular Ca2+, CaMKII inhibition of AC3 may contribute to termination of olfactory signaling. To test this hypothesis, we generated a polyclonal antibody specific for AC3 phosphorylated at Ser-1076. A brief exposure of mouse olfactory cilia or primary olfactory neurons to odorants stimulated phosphorylation of AC3 at Ser-1076. This phosphorylation was blocked by inhibitors of CaMKII, which also ablated cAMP decreases associated with odorant-stimulated cAMP transients. These data define a novel mechanism for termination of olfactory signaling that may be important in olfactory responses.

Leptin inhibits insulin secretion by activation of phosphodiesterase 3B.

The molecular signaling events by which leptin exerts its functions in vivo are not well delineated. Here, we show a novel leptin signaling mechanism that requires phosphoinositide 3-kinase (PI 3-kinase)-dependent activation of cyclic nucleotide phosphodiesterase 3B (PDE3B) and subsequent suppression of cAMP levels. In pancreatic beta cells, leptin causes the activation of PDE3B, which leads to marked inhibition of glucagon-like peptide-1-stimulated insulin secretion. The effect of leptin is abolished when insulin secretion is induced with cAMP analogues that cannot be hydrolyzed by PDE3B. Selective inhibitors of PDE3B and PI 3-kinase completely prevent the leptin effect on insulin secretion and cAMP accumulation. The results demonstrate that one of the physiological effects of leptin, suppression of insulin secretion, is mediated through activation of PDE3B and suggest PDE3B as a mediator of leptin action in other tissues.

Attenuation of insulin secretion by insulin-like growth factor 1 is mediated through activation of phosphodiesterase 3B.

Both insulin and insulin-like growth factor 1 (IGF-1) are known to reduce glucose-dependent insulin secretion from the beta cells of pancreatic islets. In this paper we show that the mechanism by which IGF-1 mediates this effect is in large part through activation of a specific cyclic nucleotide phosphodiesterase, phosphodiesterase 3B (PDE3B). More specifically, in both isolated pancreatic islets and insulin-secreting HIT-T15 cells, IGF-1 inhibits insulin secretion that has been increased by glucose and glucagonlike peptide 1 (GLP-1). Moreover, IGF-1 decreases cAMP levels in parallel to the reduction of insulin secretion. Insulin secretion stimulated by cAMP analogs that activate protein kinase A and also are substrates for PDE3B is also inhibited by IGF-1. However, IGF-1 does not inhibit insulin secretion stimulated by nonhydrolyzable cAMP analogs. In addition, selective inhibitors of PDE3B completely block the ability of IGF-1 to inhibit insulin secretion. Finally, PDE3B activity measured in vitro after immunoprecipitation from cells treated with IGF-1 is higher than the activity from control cells. Taken together with the fact that pancreatic beta cells express little or no insulin receptor but large amounts of IGF-1 receptor, these data strongly suggest a new regulatory feedback loop model for the control of insulin secretion. In this model, increased insulin secretion in vivo will stimulate IGF-1 synthesis by the liver, and the secreted IGF-1 in turn feedback inhibits insulin secretion from the beta cells through an IGF-1 receptor-mediated pathway. This pathway is likely to be particularly important when levels of both glucose and secretagogues such as GLP-1 are elevated.

A subset of olfactory neurons that selectively express cGMP-stimulated phosphodiesterase (PDE2) and guanylyl cyclase-D define a unique olfactory signal transduction pathway.

Odorant information is encoded by a series of intracellular signal transduction events thought to be mediated primarily by the second messenger cAMP. We have found a subset of olfactory neurons that express the cGMP-stimulated phosphodiesterase (PDE2) and guanylyl cyclase-D (GC-D), suggesting that cGMP in these neurons also can have an important regulatory function in olfactory signaling. PDE2 and GC-D are both expressed in olfactory cilia where odorant signaling is initiated; however, only PDE2 is expressed in axons. In contrast to most other olfactory neurons, these neurons appear to project to a distinct group of glomeruli in the olfactory bulb that are similar to the subset that have been termed "necklace glomeruli." Furthermore, this subset of neurons are unique in that they do not contain several of the previously identified components of olfactory signal transduction cascades involving cAMP and calcium, including a calcium/calmodulin-dependent PDE (PDE1C2), adenylyl cyclase III, and cAMP-specific PDE (PDE4A). Interestingly, these latter three proteins are expressed in the same neurons; however, their subcellular distribution is distinct. PDE1C2 and adenylyl cyclase III are expressed almost exclusively in the olfactory cilia whereas PDE4A is present only in the cell bodies and axons. These data strongly suggest that selective compartmentalization of different PDEs and cyclases is an important feature for the regulation of signal transduction in olfactory neurons and likely in other neurons as well. In addition, the data implies that an olfactory signal transduction pathway specifically modulated by cGMP is present in some neurons of the olfactory neuroepithelium.

SpHbox7, a new Abd-B class homeobox gene from the sea urchin Strongylocentrotus purpuratus: insights into the evolution of hox gene expression and function.

Hox genes, by virtue of their key functions in axial patterning, have long been thought to be pivotal players in the evolution of developmental mechanisms. Despite their potential importance in evolution, there is little information about Hox genes in animal groups that are most closely related to ancestral Chordates. Accordingly, we have taken the step of analyzing Hox gene expression and function in the sea urchin embryo, whose simple bilateral body plan is thought to resemble that of a stem organism in the Chordate lineage. Here we describe the isolation, sequences analysis and spatiotemporal expression pattern of a sea urchin (Strongylocentrotus purpuratus) Abd-B-like gene, designated SpHbox7. We show that this gene is one of at least two Abd-B-like genes in the S. purpuratus genome, a result that argues against the simple hypothesis that Hox gene duplications occurred only during the evolution of the chordates. SpHbox7 transcripts are first detectable in midblastula stage embryos, increase in amount during gastrulation, decline slightly by the pluteus stage, and are not detectable in any tissue of the adult. Whole mount in situ hybridization and antibody staining with an SpHbox7-specific antibody reveal that both SpHbox7 mRNA and protein are present throughout the embryo in the blastula. Subsequently, they are localized in the invaginating archenteron, secondary mesenchyme, and oral ectoderm. By the pluteus larva stage, SpHbox7 protein and mRNA are present in the gut, larval arms, and portions of the oral ectoderm. This complex and dynamic expression pattern suggests that SpHbox7 has a role in the patterning of the gut, the mesoderm, and the oral surface.

The calmodulin-dependent phosphodiesterase gene PDE1C encodes several functionally different splice variants in a tissue-specific manner.

We report here the identification of cDNAs for three new mouse PDE1C splice variants and the characterization of their kinetics, regulation by Ca2+, sensitivities to inhibitors, and tissue/cellular expression patterns. Sequence analysis indicated that these three cDNAs (PDE1C1, PDE1C4, and PDE1C5), together with our previously reported PDE1C2 and PDE1C3, are alternative splice products of the PDE1C gene. The results from RNase protection analysis and in situ hybridization indicated that the expression of the different PDE1C splice variants is differentially regulated in a tissue/cell-specific manner. Particularly, high levels of PDE1C mRNAs were found in the olfactory epithelium, testis, and several regions of mouse brain such as cerebellar granule cells. All of these splice variants have similar kinetic properties, showing high affinities and approximately the same relative Vmax values for both cAMP and cGMP. However, they responded to Ca2+ stimulation differently. In addition, they show different sensitivities to the calmodulin-dependent phosphodiesterase inhibitors, KS505a and SCH51866. Substrate competition experiments suggested the presence of only one catalytic site on these PDE1C isozymes for both cAMP and cGMP. In summary, these findings suggest that the PDE1C gene undergoes tissue-specific alternative splicing that generates structurally and functionally diverse gene products.

Molecular cloning and characterization of a calmodulin-dependent phosphodiesterase enriched in olfactory sensory neurons.

The sensing of an odorant by an animal must be a rapid but transient process, requiring an instant response and also a speedy termination of the signal. Previous biochemical and electrophysiological studies suggest that one or more phosphodiesterases (PDEs) may play an essential role in the rapid termination of the odorant-induced cAMP signal. Here we report the molecular cloning, expression, and characterization of a cDNA from rat olfactory epithelium that encodes a member of the calmodulin-dependent PDE family designated as PDE1C. This enzyme shows high affinity for cAMP and cGMP, having a Km for cAMP much lower than that of any other neuronal Ca2+/calmodulin-dependent PDE. The mRNA encoding this enzyme is highly enriched in olfactory epithelium and is not detected in six other tissues tested. However, RNase protection analyses indicate that other alternative splice variants related to this enzyme are expressed in several other tissues. Within the olfactory epithelium, this enzyme appears to be expressed exclusively in the sensory neurons. The high affinity for cAMP of this Ca2+/calmodulin-dependent PDE and the fact that its mRNA is highly concentrated in olfactory sensory neurons suggest an important role for it in a Ca(2+)-regulated olfactory signal termination.

Activation of the L1 late H2B histone gene in blastula-stage sea urchin embryos by Antennapedia-class homeoprotein.

The L1 late H2B histone gene of the sea urchin Strongylocentrotus purpuratus is transcriptionally activated in late blastula stage embryos by a mechanism that depends on an enhancer element located 3' of the gene (Zhao et al., 1990). A protein factor, designated H2B abp 1, binds this element at a site that resembles the consensus recognition sequence of Antennapedia-class homeodomain proteins. We demonstrate here that Antennapedia (Antp) and Hbox4 proteins, members of the Antennapedia class of homeoproteins from Drosophila and sea urchin respectively, bind the L1 H2B abp 1 site, and that the Drosophila Antp protein acts through this site to trans-activate the L1 H2B gene, in vivo. In addition, RNA gel blot analysis demonstrated that Hbox4 transcripts accumulate in developing embryos with a time course that closely resembles that of H2B adp 1 DNA binding activity and the activity and the transcription rate of the L1 late H2B gene. Finally, we show that antibody prepared against the sea urchin Hbox4 protein, a member of the Abd-B subclass of the Antennapedia class, specifically inhibits binding of the H2B abp 1 factor to the L1 H2B enhancer, suggesting that H2B abp 1 is encoded by Hbox4 or a closely related gene.

Activation of a late H2B histone gene in blastula-stage sea urchin embryos by an unusual enhancer element located 3' of the gene.

In the sea urchin embryo, late histone genes are transcribed at low levels during cleavage and blastula formation and at substantially higher levels in later stages of embryogenesis. To investigate the molecular basis of the stage-specific expression of a late H2B histone gene, we injected mutant genes lacking portions of 5'- and 3'-flanking regions into Lytechinus pictus embryos and monitored their expression by RNase protection. A 200-bp region located 489 bp downstream of the mRNA 3' terminus was necessary for the increase in transcription of the late H2B gene at the mid-blastula stage of development. DNase I and methylation interference footprint analyses located only one factor-binding site in this region, and gel mobility shift experiments showed that the DNA-binding activity of this factor (designated H2B abp 1) paralleled the transcriptional activity of the L1 H2B gene. Additional mutagenesis and microinjection experiments located the activator element to a 32-bp DNA segment that includes the H2B abp 1-binding site. These experiments also showed that the 32-bp fragment functions independently of position and orientation and therefore has the hallmarks of an enhancer. That this fragment contains most or all of the L1 H2B gene transcription-stimulatory activity makes it unusual among enhancerlike elements, which generally consist of several clustered factor-binding sites that act additively or cooperatively to affect transcription. The nucleotide sequence of the L1 H2B enhancer element suggests that the trans-acting factor that interacts with it is a member of the antennapedia or engrailed class of homeodomain proteins.