Immunocytochemical detection of the intranuclear variations of phosphatidylinositol 4,5-bisphosphate amount associated with changes of activity and amount of phospholipase C β1 in cells exposed to mitogenic or differentiating agonists

The intracellular localizations of phosphatidylinositol 4,5-bisphosphate (PIP₂) and of its hydrolyzing enzyme phospholipase C (PLC; in this case the β₁ isoform) have been evaluated by electron microscope immunocytochemistry in cells exposed to mitogenic or differentiating agents. These cells have been previously demonstrated to present a signal transduction system based on the polyphosphoinositide hydrolysis localized at the nuclear level, which can be specifically modulated by agonists. The results demonstrate that in Swiss 3T3 mouse fibroblasts mitogenically stimulated by insulin-like growth factor I (IGF-I), a rapid and transient decrease of the PIP₂ detectable by immunogold labeling occurs at the nuclear interior. This effect appears due to the activation of the PLC β₁ isozyme already present in the nucleus, since no significant variations of the enzyme amount and distribution can be detected by immunolabeling. However, after 30 min of exposure to IGF-I, when the PLC β₁ activity is returned to basal level, a slight but significant increase of the enzyme amount is detected both in the nucleus and in the cytoplasm. On the other hand, an increased accumulation of PIP₂ in the nucleus, accompanied by a decrease of the intranuclear amount of PLC β₁ isozyme, have been observed in mouse erythroleukemia Friend cells, induced to erythroid differentiation by dimethylsulfoxide (DMSO). These results indicate that quantitative immunocytochemistry represents an increment in the available methodologies to investigate the complex regulation of nuclear PI-signalling.     

Structural and immunochemical characterization of β-1,2-linked mannobiosyl phosphate residue in the cell wall mannan of Candida glabrata

A mannan of Candida glabrata IFO 0622 digested by Arthrobacter exo-α-mannosidase and a β-1,2-linked mannobiose obtained from the parent mannan by acid treatment was analyzed using ¹³C nuclear magnetic resonance spectroscopy. The results show that the β-1,2-linked mannobiosyl residue is esterified to a phosphate group through position C-1 in the α-configuration, Manβ1– 2Manα1–HPO₃–. The results of immunochemical assays of these mannans using the commercial antigenic factor sera of the genus Candida (Candida Check, Iatron) indicate that the main recognition site of serum no. 6 in this kit is the mannotetraosyl side-chain Manβ1–2Manα1– 2Manα1–2Man in C. glabrata mannan and also suggest that the phosphate-containing unit (such as Manβ1– 2Manα1–HPO₃– in this mannan) behaves as one of the antigenic determinants of serum no. 6, but not of serum no. 5. Therefore, the present and previous findings indicate that serum no. 5 recognizes relatively longer β-1,2-linked oligomannosyl side-chains, Manβ1–[2Manβ1–]_n 2Man (n = 1–6), attached to the phosphate groups previously observed in the cell wall mannans of Candida albicans, Candida stellatoidea, and Candida tropicalis. Received: 18 March 1997 / Accepted: 16 September 1997     

Hypertonicity activates GSK3β in tumor cells

In this study, we examined changes in the biochemical and inotropic events of the α₁-adrenoceptor signaling pathway in hypothyroid rat hearts. Hypothyroidism was induced by treating experimental animals with 0.05% 6-n-propyl-2-thiouracil (PTU) in drinking water for 7 weeks. A significant decrease of β- and an increase in α₁-adrenoceptor density as well as an increase in the basal activity of the phosphoinositide (4,5) bisphosphate hydrolyzing phospholipase C was observed in sarcolemmal membranes purified from hypothyroid hearts as compared to age-matched euthyroid controls. Following stimulation with 10 μM phenylephrine (in the presence of 10 μM atenolol), the increase of contractile parameters over baseline values was significantly higher in hypo- than euthyroid hearts, while the opposite occurred under β-stimulation with 0.1 μM isoproterenol. Interestingly, the increase in phenylephrine-mediated positive inotropy was accompanied by a significant increase in the sarcolemmal phospholipase C activity and in the inositol 1,4,5-trisphosphate content in hypothyroid as compared to euthyroid controls. Our results suggest that cardiac α₁-adrenoceptor and its associated phosphoinositide signaling pathway may act as a reserve for catecholamine inotropic response in hypothyroidism, where the β-adrenoceptors are compromised. Deceased     

The Role of TNF-<Emphasis Type="Italic">α</Emphasis> and its Receptors in the Production of <Emphasis Type="Italic">β</Emphasis>-1,4-galactosyltransferase I mRNA by Rat Primary Type-2 Astrocytes

β-1,4-galactosyltransferase I (β-1,4-GalT I) plays an important role in the synthesis of the backbone structure of adhesion molecules involved in leukocyte–endothelial cell interaction. The expression of β-1,4-GalT I mRNA increased in primary human endothelial cells after exposure to tumor necrosis factor-α (TNF-α). In the central nervous system (CNS), astrocytes play a pivotal role in immunity as immunocompetent cells by secreting cytokines and inflammatory mediators, there are two types of astrocytes. Type-1 astrocytes can secrete TNF-α when stimulated with Lipopolysaccharide (LPS), while the responses of type-2 astrocytes during inflammation are unknown. So we examined the expression change of β-1,4-GalT I mRNA in type-2 astrocytes after exposure to TNF-α and LPS. Real-time PCR showed that TNF-α or LPS affected β-1,4-GalT I mRNA expression in a time- and dose-dependent manner. RT-PCR analysis revealed that TNFR1 and TNFR2 were present in normal untreated type-2 astrocytes, and that TNF-α, TNFR1 and TNFR2 increased in type-2 astrocytes after exposure to TNF-α or LPS. Immunocytochemistry showed that TNFR1 was expressed in the cytoplasm, nucleus and processes of normal untreated type-2 astrocytes, and distributed mainly in the cytoplasm and processes after exposure to LPS. TNFR2 was mainly expressed in the nucleus of normal untreated type-2 astrocytes, and distributed mainly in the processes of type-2 astrocytes after exposure to LPS. Both anti-TNFR1 and anti-TNFR2 antibodies suppressed β-1,4-GalT I mRNA expression induced by TNF-α or LPS. From these results, we conclude that TNF-α signaling via both TNFR1 and TNFR2 translocated from nucleus to cytoplasm or processes is sufficient to induce β-1,4-GalT I mRNA. In addition, we observed that not only exogenous TNF-α but also TNF-α produced by type-2 astrocytes affected β-1,4-GalT I mRNA production in type-2 astrocytes. These results suggest that an autocrine loop involving TNF-α contributes to the production of β-1,4-GalT I mRNA in response to inflammation. Chunlin Xia is the co-first author.     

Physiological significance of P2X receptor-mediated vasoconstriction in five different types of arteries in rats

P2X₁ receptors, the major subtype of P2X receptors in the vascular smooth muscle, are essential for α,β-methylene adenosine 5′-triphosphate (α,β-MeATP)-induced vasoconstriction. However, relative physiological significance of P2X₁ receptor-regulated vasoconstriction in the different types of arteries in the rat is not clear as compared with α₁-adrenoceptor-regulated vasoconstriction. In the present study, we found that vasoconstrictive responses to noncumulative administration of α,β-MeATP in the rat isolated mesenteric arteries were significantly smaller than those to single concentration administration of α,β-MeATP. Therefore, we firstly reported the characteristic of α,β-MeATP-regulated vasoconstrictions in rat tail, internal carotid, pulmonary, mesenteric arteries, and aorta using single concentration administration of α,β-MeATP. The rank order of maximal vasoconstrictions for α,β-MeATP (E _{max·α,β-MeATP}) was the same as that of maximal vasoconstrictions for noradrenaline (E _max·NA) in the internal carotid, pulmonary, mesenteric arteries, and aorta. Moreover, the value of (E _{max·α,β-MeATP}/E _max·KCl)/(E _max·NA/E _max·KCl) was 0.4 in each of the four arteries, but it was 0.8 in the tail artery. In conclusion, P2X₁ receptor-mediated vasoconstrictions are equally important in rat internal carotid, pulmonary, mesenteric arteries, and aorta, but much greater in the tail artery, suggesting its special role in physiological function.     

Subunit Regulation of the Human Brain α1E Calcium Channel

The α₁ subunit coding for the human brain type E calcium channel (Schneider et al., 1994) was expressed in Xenopus oocytes in the absence, and in combination with auxiliary α₂δ and β subunits. α_1E channels directed with the expression of Ba²⁺ whole-cell currents that completely inactivated after a 2-sec membrane pulse. Coexpression of α_1E with α_2bδ shifted the peak current by +10 mV but had no significant effect on whole-cell current inactivation. Coexpression of α_1E with β_2a shifted the peak current relationship by −10 mV, and strongly reduced Ba²⁺ current inactivation. This slower rate of inactivation explains that a sizable fraction (40 ± 10%, n= 8) of the Ba²⁺ current failed to inactivate completely after a 5-sec prepulse. Coinjection with both the cardiac/brain β_2a and the neuronal α_2bδ subunits increased by ≈10-fold whole-cell Ba²⁺ currents although coinjection with either β_2a or α_2bδ alone failed to significantly increase α_1E peak currents. Coexpression with β_2a and α_2bδ yielded Ba²⁺ currents with inactivation kinetics similar to the β_2a induced currents, indicating that the neuronal α_2bδ subunit has little effect on α_1E inactivation kinetics. The subunit specificity of the changes in current properties were analyzed for all four β subunit genes. The slower inactivation was unique to α_1E/β_2a currents. Coexpression with β_1a, β_1b, β₃, and β₄, yielded faster-inactivating Ba²⁺ currents than currents recorded from the α_1E subunit alone. Furthermore, α_1E/α_2bδ/β_1a; α_1E/α_2bδ/β_1b; α_1E/α_2bδ/β₃; α_1E/α_2bδ/β₄ channels elicited whole-cell currents with steady-state inactivation curves shifted in the hyperpolarized direction. The β subunit-induced changes in the properties of α_1E channel were comparable to modulation effects reported for α_1C and α_1A channels with β₃≈β_1b > β_1a≈β₄≫β_2a inducing fastest to slowest rate of whole-cell inactivation. Received: 27 March 1997/Revised: 10 July 1997     

Yeast surviving factor Svf1 as a new interacting partner, regulator and in vitro substrate of protein kinase CK2

Since Svf1 is phosphoprotein, we investigated whether it was a substrate for protein kinase CK2. According to the amino acid sequence Svf1 harbours 20 putative CK2 phosphorylation sites. Here, we have reported cloning, overexpression, purification and characterization of yeast Svf1 as a substrate for three forms of yeast CK2. Svf1 serves as a substrate for both the recombinant CK2α (K _m 0.35 μM) and CK2α′ (K _m 0.18 μM) as well as CK2 holoenzyme (K _m 1.1 μM). Different K _m values argue that CK2β(β′) subunit has an inhibitory effect on the activity of both CK2α and CK2α′ towards surviving factor Svf1. Reconstitution of α′₂ββ′ isoform of CK2 holoenzyme shows that β/β′ subunits have regulatory effect depending on the kind of CK2 catalytic subunit. This effect was not observed in the case of α₂ββ′ isoform, which may be due to interaction between Svf1 and regulatory CK2β subunit (shown by co-immunoprecipitation experiments). Interactions between CK2 subunits and Svf1 protein may have influence on ATP as well as ATP-competitive inhibitors (TBBt and TBBz) binding. CK2 phosphorylates up to six serine residues in highly acidic peptide K¹⁹⁹EVIPESDEEESSADEDDNEDEDEESGDSEEESGSEEESDSEEVEITYED²⁴⁸ of the Svf1 protein in vitro. Presented data may help to elucidate the role of protein kinase CK2 and Svf1 in the regulation of cell survival pathways.     

Developmental changes in ventricular myosin isoenzymes of the tammar wallaby

Ventricular myosin in eutherian mammals undergoes a perinatal change in response to a sharp rise in thyroid hormone levels during development. In this investigation, changes in ventricular myosin heavy chains (MyHCs) of the tammar wallaby (Macropus eugenii) from early pouch life to adulthood were analysed using native gel electrophoresis, SDS-PAGE and western blotting. Adult wallaby ventricle showed three myosin isoenzymes, V₁, V₂ and V₃; western blots using specific anti-α-MyHC and anti-β-MyHC antibodies showed their MyHC compositions to be αα, αβ and ββ, respectively. Ventricular muscle in early pouch joeys expressed predominantly β-MyHC. Up to 200 days, the time of initial pouch exit, α-MyHC content was around 5%. Thereafter, there was a sharp increase of α-MyHC expression to 35% by 242 days of age, eventually falling back to 23% in the adult. These changes correlate with known surges in plasma levels of thyroid hormones around pouch exit. The results suggest that ventricular myosins in a marsupial mammal also undergo a developmental change, and that marsupial ventricular myosins are thyroid responsive as in eutherians. The increased α-MyHC expression empowers the heart to meet the enhanced cardiovascular demands of out-of-pouch activity and the thermogenic action of thyroid hormones.     

Impact of heat-inactivated <Emphasis Type="Italic">Lactobacillus casei</Emphasis> and <Emphasis Type="Italic">Lactobacillus paracasei</Emphasis> strains on cytokine responses in whole blood cell cultures of children with atopic dermatitis

Heat-inactivated Lactobacillus casei LOCK 0900, L. casei LOCK 0908 and Lactobacillus paracasei LOCK 0919 strains, applied to blood cell cultures obtained from children with atopic dermatitis induced production of anti-allergic T_H1 cytokines (interleukin-12, interleukin-18, interferon-γ, tumor necrosis factor-α) and regulatory transforming growth factor-β₁), but did not stimulate pro-allergic interleukin-5. The lactobacilli-mixture remarkably enhanced the T_H1 response compared to single strains. This synergistic effect was not observed for transforming growth factor-β₁. In contrast, the amount of interleukin-10 was found to be considerably lower when cells were stimulated with lactobacilli-mixture compared to single strains. The mixture of Lactobacillus strains represents a probiotic bacterial preparation modulating in vitro cytokine profile of allergic children towards anti-allergic T_H1 response.     

Simultaneous α/β spin-state selection for <Superscript>13</Superscript>C and <Superscript>15</Superscript>N from a time-shared HSQC-IPAP experiment

Two novel HSQC-IPAP approaches are proposed to achieve α/β spin-state editing simultaneously for ¹³C and ¹⁵N in a single NMR experiment. The pulse schemes are based on a time-shared (TS) 2D ¹H,¹³C/¹H,¹⁵N-HSQC correlation experiment that combines concatenated echo elements for simultaneous J(CH) and J(NH) coupling constants evolution, TS evolution of ¹³C and ¹⁵N chemical shifts in the indirect dimension and heteronuclear α/β-spin-state selection by means of the IPAP principle. Heteronuclear α/β-editing for all CH _n (n = 1–3) and NH _n (1–2) multiplicities can be achieved in the detected F2 dimension of a single TS-HSQC-F2-IPAP experiment. On the other hand, an alternative TS-HSQC-F1-IPAP experiment is also proposed to achieve α/β-editing in the indirect F1 dimension. Experimental and simulated data is provided to evaluate these principles in terms of sensitivity and performance simultaneously on backbone and side-chain CH, CH₂, CH₃, NH, and NH₂ spin systems in uniformly ¹³C/¹⁵N-labeled proteins and in small natural-abundance peptides.