G protein-coupled receptors in and on the cell nucleus: a new signaling paradigm?

Signaling from internalizing and endosomal receptors has almost become a classic GPCR paradigm in the last several years. However, it has become clear in recent years that GPCRs also elicit signals when resident at other subcellular sites including the endoplasmic reticulum, Golgi apparatus, and the nucleus. In this review we discuss the nature, function, and trafficking of nuclear GPCR signaling complexes, as well as potential sources of endogenous and exogenous ligands. Finally, we pose a series of questions that will need to be answered in the coming years to confirm and extend this as a new paradigm for GPCR signaling.     

Actin in the nucleus: what form and what for?

Actin is an abundant protein in most nonmuscle cells. It has often been observed in isolated nuclei, yet cytoplasmic contamination was of course initially regarded as the most plausible origin. Numerous studies on nuclear actin appeared in the 1970s and 1980s, but the picture remained rather muddy. The viewpoint at that time was that actin-shown to move freely between cytoplasm and nucleus-was a mere "thermodynamic wanderer," transiently occupying the nucleus. More recently, evidence has been mounting that actin's presence in the nucleus is not simply governed by the laws of diffusion. The same holds true for the finding of various actin-related proteins in the nucleus, and the case for nuclear myosin, specifically myosin I, is now quite convincing. Moreover, the first intimations of functional roles of nuclear actin are now emerging. Here we examine the overall subject from cell biological and chemical perspectives. The major issue is no longer the presence of actin in the nucleus but rather its supramolecular organization, intranuclear locations, and, of course, functions. These issues interface with recent findings that reveal a surprisingly diverse repertoire of actin conformations and oligomer and polymer forms beyond monomeric G-actin and polymeric F-actin. We present ideas for advancing the nuclear actin field and call for a renewed attack on this major problem in cell biology.     

Arthropodization in the Hirudinea: evidence for a phylogenetic link with insects and other Uniramia?

The Hirudinea have a number of arthropod-like characters, including a true haemocoel and compound eyes. This 'arthropodization' may have bearing on the origin of the arthropod phylum Uniramia. Certain traits shared by Clitellata and Uniramia are interpreted as primitive and compatible with the view that a monophyletic link exists between these two groups: 'internal' fertilization, egg protected by cocoon/chorion, egg yolky with direct development not invoking a planktotrophic larval stage, formation and fate of presumptive areas in early embryology, uniramous lobopodial-like structures and the presence of uniramous mouthparts. Still other traits shared by Hirudinea and Hexapoda are interpreted as advanced and evidence for a paraphyletic relationship between leeches and pterygote insects: oogenesis with nurse cells, cephalization and segmental constancy. It is proposed that a major theme in uniramian evolution is the invasion of land from freshwater by a pre-lobopodial clitellate-like ancestor, the lobopodium being an adaptation to terrestrial locomotion. These views give new significance to the poorly studied fossil segmented worms from freshwater and terrestrial palaeohabitats.     

Ammonium nutrition in the halophyte Spartina alterniflora under salt stress: evidence for a priming effect of ammonium?

Background and aims

The effects of salt stress on the salt marsh halophyte Spartina alterniflora have been well documented. However, plant responses to combined salinity and ammonium toxicity and the underlying mechanisms are relatively unknown. The aim of the present investigation was to study the effects of both salinity (0, 200 and 500 mM NaCl) and nitrogen form (NO₃ ^?, NH₄ ⁺ or NH₄NO₃) on S. alterniflora.

Methods

Plants were cultivated in sandy soil under greenhouse conditions for 3 months. At harvest, growth parameters were measured and leaf samples were analysed for oxidative stress parameters (malondialdehyde, MDA; electrolyte leakage, EL; and hydrogen peroxide, H₂O₂ concentration) and the activity of antioxidant enzymes (glutathione reductase, GR; superoxide dismutase, SOD; catalase, CAT; ascorbate peroxidase, APX and Guaiacol peroxidase, GPX).

Results

In the absence of NaCl, plant growth rate was the highest in the medium containing both nitrogen forms, and the lowest in the medium containing only nitrate. Irrespective of the nitrogen form, plant growth was generally higher at 200 mM NaCl than without salinity. Ammonium-fed plants showed better growth than nitrate-fed plants under high salinity. In the absence of salinity, ammonium-fed plants showed higher SOD, APX, GR, CAT, and GPX activities than nitrate-fed ones. The antioxidant enzymes exhibited higher activity in saline-treated plants. The considerable advantage of NH₄ ⁺ nutrition to S. alterniflora under saline conditions was associated with high antioxidant enzyme activities, together with low MDA content, EL, and H₂O₂ concentration.

Conclusion

These data clearly demonstrate that NH₄ ⁺ is more favourable for the growth of S. alterniflora under high salinity than NO₃ ^?. It is suggested that NH₄ ⁺ nutrition improves the plant’s capacity to limit oxidative damage by stimulating the activities of the major antioxidant enzymes.     

Anomalous effect of uncouplers on respiratory chain-linked transhydrogenation in Escherichia coli membranes: evidence for a localized proton pathway?

Energization of the pyridine nucleotide transhydrogenase in everted membrane vesicles from Escherichia coli JM83 was compared with the process in vesicles of the same strain transformed with the plasmid pDC21 overexpressing this enzyme. Proton translocation was assayed by the quenching of the fluorescence of the probe quinacrine. Agents able to discharge transmembrane proton gradients such as nigericin and the uncouplers 3,3',4',5-tetrachlorosalicylanilide and carbonyl cyanide m-chlorophenylhydrazone inhibited ATP-dependent transhydrogenation of NADP by NADH and discharged transmembrane proton gradients generated by transhydrogenation of AcNAD by NADPH, by oxidation of NADH, and by hydrolysis of ATP. This was observed in everted membrane vesicles of both strains JM83 and JM83pDC21. These strains differed significantly in the response of the NADH oxidation-dependent transhydrogenase. This reaction was inhibited by nigericin and uncouplers in membrane vesicles of JM83 but there was little inhibition or the reaction was stimulated in JM83pDC21, in spite of the discharge of the NADH oxidation-generated proton gradient measured by quinacrine fluorescence in the latter strain. It is proposed that the transhydrogenase is energized by direct or local (nonbulk phase) proton translocation in membranes of this strain. Uncouplers might facilitate these routes but would not discharge them. The generality of these observations was shown using other strains. NADH oxidase activity was severalfold lower in membrane vesicles of JM83pDC21 compared with JM83. The levels of ubiquinone and cytochromes, and the activities of NADH dehydrogenases I and II, and of cytochrome oxidase, were similar in the two strains. It is concluded that the NADH oxidase activity of JM83pDC21 is low because of the reduced rate of collision between electron-transferring complexes of the respiratory chain due to the large amount of transhydrogenase protein in the membranes of this strain. The large amount of transhydrogenase favors direct, nonbulk phase proton transfer. Transhydrogenase activity was stimulated by Ca2+, Mg2+, or Mn2+.     

Does angiotensin II have a significant tonic action on cardiovascular neurons in the rostral and caudal VLM?

The peptidic ANG II receptor antagonists [Sar(1),Ile(8)]ANG II (sarile) or [Sar(1),Thr(8)]ANG II (sarthran) are known to decrease arterial pressure and sympathetic activity when injected into the rostral part of the ventrolateral medulla (VLM). In anesthetized rabbits and rats, the profound depressor and sympathoinhibitory response after bilateral microinjections of sarile or sarthran into the rostral VLM was unchanged after prior selective blockade of angiotensin type 1 (AT(1)) and ANG-(1---7) receptors, although this abolished the effects of exogenous ANG II. Unlike the neuroinhibitory compounds muscimol or lignocaine, microinjections of sarile in the rostral VLM did not affect respiratory activity. Sarile or sarthran in the caudal VLM resulted in a large pressor and sympathoexcitatory response, which was also unaffected by prior blockade of AT(1) and ANG-(1---7) receptors. The results indicate that the peptidic ANG receptor antagonists profoundly inhibit the tonic activity of cardiovascular but not respiratory neurons in the VLM and that these effects are independent of ANG II or ANG-(1---7) receptors.     

The protective effect of crocin on the amyloid fibril formation of aβ42 peptide in vitro

Aβ is the main constituent of the amyloid plaque found in the brains of patients with Alzheimer’s disease. There are two common isoforms of Aβ: the more common form, Aβ₄₀, and the less common but more amyloidogenic form, Aβ₄₂. Crocin is a carotenoid from the stigma of the saffron flower and it has many medicinal properties, including antioxidant effects. In this study, we examined the potential of crocin as a drug candidate against Aβ₄₂ amyloid formation. The thioflavin T-binding assay and electron microscopy were used to examine the effects of crocin on the extension and disruption of Aβ₄₂ amyloids. To further investigate the relationship between crocin and Aβ₄₂ structure, we analyzed peptide conformation using the ANS-binding assay and circular dichroism (CD) spectroscopy. An increase in the thioflavin T fluorescence intensity upon incubation revealed amyloid formation in Aβ₄₂. It was found that crocin has the ability to prevent amyloid formation by decreasing the fluorescence intensity. Electron microscopy data also indicated that crocin decreased the amyloid fibril content of Aβ. The ANS-binding assay showed that crocin decreased the hydrophobic area in incubated Aβ₄₂. CD spectroscopy results also showed that the peptide undergoes a structural change to α-helical and β-turn. Our study shows that the anti-amyloidogenic effect of crocin may be exerted not only by the inhibition of Aβ amyloid formation but also by the disruption of amyloid aggregates. Therefore, crocin could be essential in the search for therapies inhibiting aggregation or disrupting aggregation.     

Study on the effect of IRE1α on cell growth and apoptosis via modulation PLK1 in ER stress response

The mammalian unfolded protein response (UPR) protects the cell against the stress of misfolded proteins in the endoplasmic reticulum (ER). Failure to adapt to ER stress causes the UPR to trigger apoptosis. Inositol-requiring enzyme-1a (IRE1a), as one of three unfolded protein sensors in UPR signaling pathways, senses ER unfolded proteins through an ER lumenal domain that becomes oligomerized during ER stress. It is known to be important for ER stress-mediated apoptosis and cell growth, but the exact molecular mechanism underlying these processes remains unexplored. In this study, we report that knockdown of IRE1a by an siRNA silencing approach enhanced, whereas its overexpression inhibited, cell proliferation in Hepatoma cells. Besides, overexpression of IRE1a induced, while its repression inhibited, ER stress-mediated apoptosis in Hepatomas cells. Furthermore, we found that overexpressed IRE1a can down-regulate Polo-like kinase 1(PLK1) from mRNA and protein two levels. IRE1a-mediated induction of apoptosis and inhibition of proliferation in response to ER stress is through downregulation PLK1, an early trigger for G2/M transition known to be participated in regulating cell proliferation and cell apoptosis. Collectively, these findings reveal a novel critical role of IRE1a in ER stress-mediated apoptosis and the molecular mechanisms involved. IRE1a may be a useful molecular target for the development of novel predictive and therapeutic strategies in cancer.     

Fire and the demography of camelthorn (Acacia erioloba Meyer) in the southern Kalahari – evidence for a bonfire effect?

Fires in arid environments are rare, so are not deemed as important as in mesic savannas. We investigated mortality and resprouting amongst camelthorn (Acacia erioloba) after two fires (at Vaalbos National Park and Susanna farm) in semi‐arid savanna near Kimberley, South Africa. Resprouting response 18 months after a fire was the greatest amongst <6.5 m high trees; extent of foliage damage by fire and bark thickness were the next best predictors of resprouting vigour amongst that size class. The largest size class (8–12 m height) of A. erioloba suffered the greatest mortality rates (40% and 83% at Vaalbos and Susanna respectively), with damage either severe or minor. We hypothesize that large tree mortality rates are partly attributable to well‐developed assemblages of flammable subcanopy plants producing a bonfire beneath trees. These mortality rates indicate that fire reduces both tree abundance and relative representation of large trees, and although able to resprout, A. erioloba is fire‐sensitive, which may explain its restriction to Kalahari sands where rainfall is less than 900 mm year^?1. Therefore, although relatively infrequent, fires shape Kalahari woodland structure, particularly as A. erioloba is long lived and slow growing. Large trees have been shown to be important to biodiversity in the southern Kalahari, so frequent fires could impact biodiversity.     

Genotype and extra-pair paternity in the house wren: a rare-male effect?

Females in socially monogamous species may select extra-pair (EP) mates to increase the heterozygosity, and hence fitness, of their offspring. We tested this hypothesis in the house wren (Troglodytes aedon), a largely monogamous songbird in which EP young are common. We typed paired males and females, nestlings, and males on neighbouring territories, at five to seven microsatellite loci over 2 years in a Wyoming, USA, population. We identified EP sires at 20 nests with EP young. In pairwise comparisons, we found no significant differences between cuckolded within-pair (WP) males and EP sires in three measures of heterozygosity (mean d2, standardized heterozygosity and internal relatedness). However, EP sires had fewer alleles that were common within the population than did the WP males they cuckolded. Nearby males who were EP sires also had fewer common alleles than did nearby males who did not sire EP young. Females in our population may be more prone to accept copulations from males with rare genotypes than from males with common genotypes. Alternatively, selection of rare-male sperm may occur within the female reproductive tract. Because mating with rare males is likely to increase offspring heterozygosity, our data suggest that EP mating may provide genetic benefits to females.     

A comparative study of the effect of 17β-estradiol and estriol on peripheral pain behavior in rats

Although estradiol has been reported to influence pain sensitivity, the role of estriol (an estradiol metabolite and another widely used female sex hormone) remains unclear. In this study, pain behavior tests, whole-cell patch clamp recording and Western blotting were used to determine whether estriol plays a role in pain signal transduction and transmission. Either systemic or local administration of 17β-estradiol produced a significant rise of mechanical pain threshold, while estriol lacked this effect in normal and ovariectomized (OVX) rats following estriol replacement. Local administration of 17β-estradiol or estriol significantly decreased ATP-induced spontaneous hind-paw withdrawal duration (PWD), which was blocked by an estrogen receptor antagonist, ICI 182, 780. However, systemic application of estriol in normal or OVX rats lacked this similar effect. In cultured dorsal root ganglion neurons, estriol attenuated α,β-methylene ATP-induced transient currents which were blocked by ICI 182, 780. In complete Freund's adjuvant treated (CFA) rats, systemic application of 17β-estradiol or estriol decreased the mechanical pain threshold significantly, but did not change the inflammatory process. Similar effects were observed after estriol replacement in OVX rats. The expression of c-fos in lumbosacral spinal cord dorsal horn (SCDH) was increased significantly by administration of 17β-estradiol but not estriol, and not by estriol replacement in OVX rats. These results suggest that 17β-estradiol but not estriol plays an anti-hyperalgesic role in physiological pain. However, both peripheral 17β-estradiol and estriol play anti-hyperalgesic roles in ATP-induced inflammatory pain. Systemic application of estriol as well as 17β-estradiol plays hyperalgesic roles in CFA-induced chronic pain.     

Cerebral artery reactivity changes during pregnancy and the postpartum period: a role in eclampsia?

Eclampsia is thought to be similar to hypertensive encephalopathy, whereby acute elevations in intravascular pressure cause forced dilatation (FD) of intrinsic myogenic tone of cerebral arteries and arterioles, decreased cerebrovascular resistance, and hyperperfusion. In the present study, we tested the hypothesis that pregnancy and/or the postpartum period predispose cerebral arteries to FD by diminishing pressure-induced myogenic activity. We compared the reactivity to pressure (myogenic activity) as well as factors that modulate the level of tone of third-order branches (<200 microm) of the posterior cerebral artery (PCA) that were isolated from nonpregnant (NP, n = 7), late-pregnant (LP, 19 days, n = 10), and postpartum (PP, 3 days, n = 8) Sprague-Dawley rats under pressurized conditions. PCAs from all groups of animals developed spontaneous tone within the myogenic pressure range (50-150 mmHg) and constricted arteries at 100 mmHg (NP, 30 +/- 3; LP, 39 +/- 4; and PP, 42 +/- 7%; P > 0.05). This level of myogenic activity was maintained in the NP arteries at all pressures; however, both LP and PP arteries dilated at considerably lower pressures compared with NP, which lowered the pressure at which FD occurred from >175 for NP to 146 +/- 6.5 mmHg for LP (P < 0.01 vs. NP) and 162 +/- 7.7 mmHg for PP (P < 0.01 vs. NP). The amount of myogenic tone was also significantly diminished at 175 mmHg compared with NP: percent tone for NP, LP, and PP animals were 35 +/- 2, 11 +/- 3 (P < 0.01 vs. NP), and 20 +/- 7% (P < 0.01 vs. NP), respectively. Inhibition of nitric oxide (NO) with 0.1 mM N(omega)-nitro-l-arginine (l-NNA) caused constriction of all vessel types that was significantly increased in the PP arteries, which demonstrates significant basal NO production. Reactivity to 5-hydroxytryptamine (serotonin) was assessed in the presence of l-NNA and indomethacin. There was a differential response to serotonin: PCAs from NP animals dilated, whereas LP and PP arteries constricted. These results suggest that both pregnancy and the postpartum period predispose the cerebral circulation to FD at lower pressures, a response that may lower cerebrovascular resistance and promote hyperperfusion when blood pressure is elevated, as occurs during eclampsia.     

The structure of the ends of α-helices in globular proteins: effect of additional hydrogen bonds and implications for helix formation

We prepared a set of about 2000 α-helices from a relational database of high-resolution three-dimensional structures of globular proteins, and identified additional main chain i ← i+3 hydrogen bonds at the ends of the helices (i.e., where the hydrogen bonding potential is not fulfilled by canonical i ← i+4 hydrogen bonds). About one-third of α-helices have such additional hydrogen bonds at the N-terminus, and more than half do so at the C-terminus. Although many of these additional hydrogen bonds at the C-terminus are associated with Schellman loops, the majority are not. We compared the dihedral angles at the termini of α-helices having or lacking the additional hydrogen bonds. Significant differences were found, especially at the C-terminus, where the dihedral angles at positions C2 and C1 in the absence of additional hydrogen bonds deviate substantially from those occurring within the α-helix. Using a novel approach we show how the structure of the C-terminus of the α-helix can emerge from that of constituent overlapping α-turns and β-turns, which individually show a variation in dihedral angles at different positions. We have also considered the direction of propagation of the α-helix using this approach. If one assumes that helices start as a single α-turn and grow by successive addition of further α-turns, the paths for growth in the N → C and C → N directions differ in a way that suggests that extension in the C → N direction is favored.     

Guanosine 3′,5′-monophosphate-dependent protein kinase from the silkmoth,Bombyx mori: Further evidence for the involvement of kinase in the phosphorylation of vitellin and the effect of phosphorylation on the susceptibility of vitellin to a cysteine proteinase in the egg

• 1.1. In the developing silkmoth eggs, a high level of activity of a cGMP-dependent protein kinase (G-kinase) is found. Vitellin (Vn) is an excellent substrate for the kinase, suggestin that the kinase may be involved in the phosphorylaiton of Vn in intact developing eggs.
• 2.2. To characterize this system in greater detail, the following experiments were performed: (a) changes in the levels of cyclic nucleotides and changes in cyclic nucleotide-dependent protein kinase activities were monitored in the eggs; (b) ³²P-o-phosphate was microinjected into eggs to demonstrate the phosphorylation of Vn in vivo; (c) to determine the role, if any, in vivo of the phosphorylation of Vn, the effect of phosphorylation on the susceptibility of Vn to a cysteine proteinase after the phosphorylation of Vn by G-kinase was studied.
• 3.3. The results revealed that the phosphorylation of Vn serves as the trigger for the proteolytic digestion of Vn during the developments, and may be able to provide the signal for the degradtion of Vn by the cysteine proteinase.

     

Co-expression of α′ and β subunits of β-conglycinin in rice seeds and its effect on the accumulation behavior of the expressed proteins

A transgenic rice that produces both the α′ and β subunits of β-conglycinin has been developed through the crossing of two types of transgenic rice. Although the accumulation level of the α′ subunit in the α′β-transgenic rice was slightly lower than that in the transgenic rice producing only the α′ subunit, the accumulation level of the β subunit in the α′β-transgenic rice was about 60% higher than that in the transgenic rice producing only the β subunit. Results from sequential extraction and gel-filtration experiments indicated that part of the β subunit formed heterotrimers with the α′ subunit in a similar manner as in soybean seeds and that the heterotrimers interacted with glutelin via cysteine residues. These results imply that the accumulation level of the β subunit in the α′β-transgenic rice increases by an indirect interaction with glutelin. Immunoelectron microscopy revealed that the α′ and β subunits are localized in a low electron-dense region of protein body-II (PB-II) and that α′ homotrimers in the α′β-transgenic rice seeds seem to accumulate outside of this low electron-dense region.     

Complex formation of the laminin-5 γ2 chain and large unspliced tenascin-C in oral squamous cell carcinoma in vitro and in situ: implications for sequential modulation of extracellular matrix in the invasive tumor front

Invasion and metastasis in oral squamous cell carcinoma (OSCC) are associated with changes in the extracellular matrix (ECM). We have previously shown an extracellular co-deposition of laminin-5 (Ln-5) and large unspliced tenascin-C (Tn-C_L) in OSCC. Using a co-culture model of hTERT-BJ1 fibroblasts and the OSCC cell line PE/CA-PJ15, we demonstrate in the present study that Ln-5 and Tn-C_L are not only co-deposited, but also form a physical complex which can be recovered by co-immunoprecipitation. In agreement with these results, examination of OSCC tissue specimens of different malignancy grade by means of confocal laser scanning microscopy revealed different patterns of Ln-5 and Tn-C_L co-localization implicating complex formation also in vivo. A ribbon like co-localization was detected in subepithelial basement membranes around well differentiated OSCC parts and tumor clusters. Furthermore, a fibrillar Ln-5 γ2 chain/Tn-C_L co-localization occurred in the carcinoma stroma beneath tumor clusters. Additionally, at the site of ruptured basement membranes there were dot or strand like co-deposits of both molecules, but co-localizations were only rarely detectable. These different patterns may reflect a sequential modulation and reorganization of the ECM in the tumor/stroma interface as it occurs in different stages of OSCC invasion.