5-Methyldeoxycytidine monophosphate deaminase and 5-methylcytidyl-DNA deaminase activities are present in human mature sperm cells

Human mature sperm cells have a high nuclease and 5-methyldeoxycytidine monophosphate (5-mdCMP) deaminase activity. The deaminase converts the nuclease degradation product 5-mdCMP into dTMP which is further cleaved into thymine and the abasic sugar-phosphate. Both 5-methylcytidine 5' and 3' monophosphates are good substrates for the deaminase. 5-methylcytidine is not a good deaminase substrate and 5-methylcytosine (5mC) is not a substrate. A purified fraction of the deaminase free of nucleases deaminates 5mC present in intact methylated double-stranded DNA. 5-mdCMP deaminase co-purifies on SDS-PAGE with dCMP deaminase and has an apparent molecular weight of 25 kDa. The enzyme requires no divalent cations and has a Km of 1.4 x 10(-7) M for 5-mdCMP and a Vmax of 7 x 10(-11) mol/h/microg protein. The possible biological implications of the deaminase's activities in the present system are discussed.     

Small RNAs are modified with N-glycans and displayed on the surface of living cells

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Ultrastructural analysis ofSpinacia oleracea sperm cells isolated from mature pollen grains

Summary In isolated condition, the sperm cells ofSpinacia oleracea are no longer arranged in pairs as in the pollen grain. The vegetative membrane, which surrounds a sperm cell pair in a mature pollen grain, is lost during the isolation procedure. The sperm cells become spherical in shape.The isolated sperm cell is surrounded by an intact plasma membrane. The heterochromatic or euchromatic sperm cell nucleus is located in the cell center. Mitochondria are round to oval and have distinct cristae. Often they are clustered in groups of 5 to 10 mitochondria. Dictyosomes are present in the cytoplasm and consist of 4 to 5 cisterns. Endoplasmatic reticulum is mostly situated at the sperm cell periphery, as single cisterns very near the plasma membrane.From diameters of sectioned sperm cells in electron micrographs, it is possible to calculate the average diameter of the whole sperm cell. This average diameter is 3.66 m with a variation of 3.0 m to 4.2 m, resulting in an average volume of 25.6 m³. The nuclear volume is 12.8 m³ (50.0% of the whole cell) and the mitochondrial volume is 0.7 m³ (2.5% of the whole cell). The frequency distribution of the isolated sperm cells diameters shows only one peak with a normal distribution, indicating that there is no dimorphism in volume.     

Olfactory receptors and odour coding

The first step of olfactory detection involves interactions between odorant molecules and neuronal protein receptors. Odour coding results from the combinatory activation of a set of receptors and rests on their clonal expression and olfactory neurone connexion, which lead to formation of a specific sensory map in the cortex. This system, sufficient to discriminate myriads of odorants with a mere 350 different receptors, allows humans to smell molecules that are not natural (new cooking flavours, synthetic chemicals...). The extreme olfactory genome diversity explains the absence of odour semantics. Olfactory receptors are also involved in cellular chemotaxis.     

Expression of Notch receptors and ligands on immature and mature T cells

Notch plays multiple roles in T cell development in the thymus and T cell differentiation in the periphery. In order to systematically examine the role of Notch in T cell biology, we determined the cell surface expression of all Notch receptors and ligands on various populations of T cells by using a panel of specific monoclonal antibodies we recently established. Notch1 and Notch3 were upregulated at double-negative (DN) 2-DN4 stages of immature thymocytes, then downregulated on mature single-positive thymocytes and peripheral T cells, but were rapidly upregulated again upon activation. Notch2 was consistently expressed on T cells while Notch4 was not. Jagged1 and Jagged2 were expressed at double-positive stage of immature T cells. Jagged2 was also inducible on mature T cells upon activation. In contrast, no Delta-like (Dll) 1 or Dll4 expression was observed on T cells. These comprehensive profiling of the expression of Notch receptors and ligands would be informative to fully understand the role of individual Notch receptors and ligands in T cell development and differentiation.     

Olfactory receptors in non-chemosensory tissues

Olfactory receptors (ORs) detect volatile chemicals that lead to the initial perception of smell in the brain. The olfactory receptor (OR) is the first protein that recognizes odorants in the olfactory signal pathway and it is present in over 1,000 genes in mice. It is also the largest member of the G protein-coupled receptors (GPCRs). Most ORs are extensively expressed in the nasal olfactory epithelium where they perform the appropriate physiological functions that fit their location. However, recent whole-genome sequencing shows that ORs have been found outside of the olfactory system, suggesting that ORs may play an important role in the ectopic expression of non-chemosensory tissues. The ectopic expressions of ORs and their physiological functions have attracted more attention recently since MOR23 and testicular hOR17-4 have been found to be involved in skeletal muscle development, regeneration, and human sperm chemotaxis, respectively. When identifying additional expression profiles and functions of ORs in non-olfactory tissues, there are limitations posed by the small number of antibodies available for similar OR genes. This review presents the results of a research series that identifies ectopic expressions and functions of ORs in non-chemosensory tissues to provide insight into future research directions. [BMB Reports 2012; 45(11): 612-622]     

Olfactory receptors: G protein-coupled receptors and beyond

Sensing the chemical environment is critical for all organisms. Diverse animals from insects to mammals utilize highly organized olfactory system to detect, encode, and process chemostimuli that may carry important information critical for health, survival, social interactions and reproduction. Therefore, for animals to properly interpret and react to their environment it is imperative that the olfactory system recognizes chemical stimuli with appropriate selectivity and sensitivity. Because olfactory receptor proteins play such an essential role in the specific recognition of diverse stimuli, understanding how they interact with and transduce their cognate ligands is a high priority. In the nearly two decades since the discovery that the mammalian odorant receptor gene family constitutes the largest group of G protein-coupled receptor (GPCR) genes, much attention has been focused on the roles of GPCRs in vertebrate and invertebrate olfaction. However, is has become clear that the 'family' of olfactory receptors is highly diverse, with roles for enzymes and ligand-gated ion channels as well as GPCRs in the primary detection of olfactory stimuli.     

Isolation of sperm cells from mature pollen grains of Spinacia oleracea L.

Summary When mature pollen grains of Spinacia oleracea were squashed in a 25% sucrose solution and subsequently centrifuged on a percoll layer, sperm cells were isolated in high numbers. All steps were carried out at 4° C. Isolated sperm cells could be kept alive for several hours.     

Ultrastructural analysis of the sperm cells of mature pollen of maize,Zea mays

Summary Ultrastructural analysis of the mature viable unhydrated pollen of maize,Zea mays from dehiscent anthers shows that the sperm cells are physically distant, each bounded by an envelope comprising their own plasma membrane and the inner plasma membrane of the vegetative cell. The chondriome is unusual in containing one or more filamentous complexes, up to 12m in length appressed to the side of the sperm nucleus. The extensions at each end of the elongate sperm cells contain longitudinally-oriented arrays of endoplasmic lamellae. In a three-dimensional reconstruction of serial thin sections, there is a long J-shaped sperm, c. 35 × 5m and up to 1m in thickness, sited within pointed evaginations of the vegetative nucleus and a second shorter sperm c. 20 × 5m and up to 3.5m in thickness.Abbreviations PA-TCH-SP periodic acid-thiocarbohydrazide-silver proteinate - DAPI 4,6-diamino-2-phenylindole - SC sperm cell - Sn sperm nucleus - Ua-Pb Uranyl acetate-lead citrate staining - ER endoplasmic reticulum     

Olfactory receptors in aquatic and terrestrial vertebrates

In species representing different levels of vertebrate evolution, olfactory receptor genes have been identified by molecular cloning techniques. Comparing the deduced amino-acid sequences revealed that the olfactory receptor gene family of Rana esculenta resembles that of Xenopus laevis, indicating that amphibians in general may comprise two classes of olfactory receptors. Whereas teleost fish, including the goldfish Carassius auratus, possess only class I receptors, the `living fossil' Latimeria chalumnae is endowed with both receptor classes; interestingly, most of the class II genes turned out to be pseudogenes. Exploring receptor genes in aquatic mammals led to the discovery of a large array of only class II receptor genes in the dolphin Stenella Coeruleoalba; however, all of these genes were found to be non-functional pseudogenes. These results support the notion that class I receptors may be specialized for detecting water-soluble odorants and class II receptors for recognizing volatile odorants. Comparing the structural features of both receptor classes from various species revealed that they differ mainly in their extracellular loop 3, which may contribute to ligand specificity. Comparing the number and diversity of olfactory receptor genes in different species provides insight into the origin and the evolution of this unique gene family. Accepted: 29 July 1998     

Cryopreservation of epididymal dog sperm.

A method of cryopreservation was developed for sperm salvaged from the cauda epididymis and vas deferens of domestic dog testes. Four modifications of the glycerol concentration of a buffer used for cryopreservation of dog ejaculates and two freezing rates were assessed for their effect upon post-thaw spermatozoal motility and morphology. There was no statistical difference between the four glycerol concentrations or the two freezing rates and the buffer containing 6% glycerol and the freezing rate provided by 0.5 ml straws was chosen for further study. This method resulted in a significant reduction in the percentage of live spermatozoa detected with Hoechst staining and a reduction in the percentage of capacitated spermatozoa after freeze-thawing. However, there was no difference in the ability of frozen-thawed spermatozoa to penetrate homologous oocytes.This study demonstrates that cryopreservation of epididymal canine sperm can be performed using methods similar to those established for ejaculates of the same species, and that despite some damage, spermatozoa retain their functional ability.     

Olfactory receptors on the antennae of the malaria mosquito Anopheles gambiae are sensitive to ammonia and other sweat-borne components

Electrophysiological studies on female An. gambiae s.s. mosquitoes revealed a receptor neuron within a subpopulation of the antennal grooved-peg sensilla sensitive to the odour of incubated sweat, but not responding to fresh sweat. This receptor neuron was sensitive to ammonia as well, a sweat-borne component which attracts female An. gambiae in a windtunnel bioassay. Neurons innervating a different subpopulation of grooved-peg sensilla did not show a response to incubated sweat. In the latter sensilla, however, one type of neuron responded to water or water containing solutions, while another receptor neuron was inhibited when stimulated with dry air, ether or ethanol. Neurons innervating sensilla trichodea, a more abundant antennal type of olfactory sensillum, did not respond to fresh or incubated sweat at the doses offered. However, receptor neurons within the sensilla trichodea responded with excitation to several sweat-borne components. A subpopulation of the sensilla trichodea was innervated by neurons sensitive to geranyl acetone. A second subpopulation housed receptor neurons sensitive to indole. 3-Methyl-1-butanol and 6-methyl-5-hepten-2-one evoked excitation of receptor neurons within both subpopulations of sensilla trichodea. Neurons were most sensitive to indole and geranyl acetone with a threshold of 0.01%. These findings are discussed in the context of host-seeking behaviour.     

Fibronectin receptors are functional on mitotic Chinese hamster ovary cells.

In this paper, evidence is provided indicating that the blockade of presynchronized CHO 15B cells in prometaphase by nocodazole is fully reversible and efficient enough to allow us to analyze the function of the integrin receptors. Flow cytometry analysis using a specific antibody raised against the fibronectin receptor, and binding studies of the radiolabeled fibronectin on the cell membrane, indicated a stable number of receptors at the cell surface during mitosis. Furthermore, in the mean time, only a slight increase in the Kd value of the fibronectin-receptor interaction was detected. A binding assay designed to test the affinity of the receptor for its extracellular ligand in an insoluble form was used. No difference was observed between mitotic and interphasic cells. Taken together, these results indicate that the rounding up of the cells observed during mitosis is not due to a loss of the receptor affinity for its extracellular ligand.     

Incomplete translocations induced in mature sperm of Drosophila

Frequencies of incomplete translocations in subcentromeric heterochromatin induced by gamma-rays in mature sperm were estimated in the "two right acrocentrics and left compound" system of Drosophila chromosome 2. The value obtained at 3000R was (2.5-2.9) X 10(-3). Apart from incomplete translocations leading to formation of new metacentrics, acrocentrics were also formed. It is suggested that they appear via reunion of fragments after replication of paternal chromosomes. Incomplete translocation were shown to be formed as a result of loss of centric heterochromatin fragment of acrocentric and acentric arm of the compound, as opposed to formation, due to loss of acentric heterochromatin fragment of acrocentric and centric fragment of the compound. Analysis of the dose-effect curve for half-translocations show that the latter are two hit events.     

Functional bitter taste receptors are expressed in brain cells

Hepatic stellate cells (HSC) store retinoids and upon activation differentiate into myofibroblast-like cells, a process whereby they lose their retinoid-containing lipid droplets. We reported earlier, activation of tissue factor (TF) in our MCT/LPS hepatotoxicity model. We now report the involvement of TF in the release of retinoid receptors RAR-α and RXR-α as accumulated lipid droplet during monocrotaline/lipopolysaccharide (MCT/LPS)-liver injury. Constitutive expression of RAR-α was observed in HSCs and endothelial cells of bile duct and portal vein, while expression of RXR-α was observed in certain pericentral hepatocytes and HSCs. Administration of sub-toxic doses of MCT or LPS strongly increased TF and RXR-α but not RAR-α expressions in HSCs and hepatocytes. However MCT/LPS co-treatment showed insoluble droplets containing RAR-α and RXR-α in the vicinity of the necrotic areas. Blocking TF with TF antisense oligonucleotides (TF-AS ODN) led to normal hepatocyte expression of RXR-α and upregulated the expression of RAR-α in HSCs. This study shows clear evidence of in vivo release of RAR-α and RXR-α as insoluble lipid droplets in liver injury. It is possible that these insoluble droplets of RAR-α and RXR-α could be used as markers for liver injury in general and activation of HSCs in particular. RXR-α appears to be a more sensitive than RAR-α as it was affected by even the subtoxic doses of MCT or LPS. The fact that TF-AS treatment not only down-regulated TF but also obliterated the release of RAR-α and RXR-α as insoluble lipid droplets in hepatocytes points towards TF being an important regulatory molecule for RAR-α and RXR-α.