Expression of barley SUSIBA2 transcription factor yields high-starch low-methane rice.

Atmospheric methane is the second most important greenhouse gas after carbon dioxide, and is responsible for about 20% of the global warming effect since pre-industrial times. Rice paddies are the largest anthropogenic methane source and produce 7-17% of atmospheric methane. Warm waterlogged soil and exuded nutrients from rice roots provide ideal conditions for methanogenesis in paddies with annual methane emissions of 25-100-million tonnes. This scenario will be exacerbated by an expansion in rice cultivation needed to meet the escalating demand for food in the coming decades. There is an urgent need to establish sustainable technologies for increasing rice production while reducing methane fluxes from rice paddies. However, ongoing efforts for methane mitigation in rice paddies are mainly based on farming practices and measures that are difficult to implement. Despite proposed strategies to increase rice productivity and reduce methane emissions, no high-starch low-methane-emission rice has been developed. Here we show that the addition of a single transcription factor gene, barley SUSIBA2 (refs 7, 8), conferred a shift of carbon flux to SUSIBA2 rice, favouring the allocation of photosynthates to aboveground biomass over allocation to roots. The altered allocation resulted in an increased biomass and starch content in the seeds and stems, and suppressed methanogenesis, possibly through a reduction in root exudates. Three-year field trials in China demonstrated that the cultivation of SUSIBA2 rice was associated with a significant reduction in methane emissions and a decrease in rhizospheric methanogen levels. SUSIBA2 rice offers a sustainable means of providing increased starch content for food production while reducing greenhouse gas emissions from rice cultivation. Approaches to increase rice productivity and reduce methane emissions as seen in SUSIBA2 rice may be particularly beneficial in a future climate with rising temperatures resulting in increased methane emissions from paddies.

Severe gastro-oesophageal reflux symptoms in relation to anxiety, depression and coping in a population-based study.

BACKGROUND: The association between psychiatric disorders and gastro-oesophageal reflux symptoms is uncertain, and few population-based studies are available. AIM: To examine the association between psychiatric and psychological factors and reflux symptoms. METHODS: Population-based, cross-sectional, case-control study based on two health surveys conducted in the Norwegian county Nord-Trondelag in 1984-1986 and 1995-1997. Reflux symptoms were assessed in the second survey, including 65,333 participants (70% of the county's adult population). 3153 subjects reporting severe reflux symptoms were defined as cases and 40,210 subjects without symptoms were defined as controls. Data were collected in questionnaires. Odds ratio with 95% confidence intervals were estimated using unconditional logistic regression, in adjusted models. RESULTS: Subjects reporting anxiety without depression had a 3.2-fold (95% CI: 2.7-3.8) increased risk of reflux, subjects with depression without anxiety had a 1.7-fold (95% CI: 1.4-2.1) increased risk and subjects with both anxiety and depression had a 2.8-fold (95% CI: 2.4-3.2) increased risk, compared to subjects without anxiety/depression. We observed a weak inverse association between one measure of covert coping and risk of reflux and a weak positive association between another coping measure and risk of reflux. CONCLUSIONS: This population-based study indicates that anxiety and depression are strongly associated with reflux symptoms, while no consistent association regarding coping and reflux was found.

Airborne occupational exposures and risk of oesophageal and cardia adenocarcinoma.

BACKGROUND: The reasons for the increasing incidence of and strong male predominance in patients with oesophageal and cardia adenocarcinoma remain unclear. The authors hypothesised that airborne occupational exposures in male dominated industries might contribute. METHODS: In a nationwide Swedish population based case control study, 189 and 262 cases of oesophageal and cardia adenocarcinoma respectively, 167 cases of oesophageal squamous cell carcinoma, and 820 frequency matched controls underwent personal interviews. Based on each study participant's lifetime occupational history the authors assessed cumulative airborne occupational exposure for 10 agents, analysed individually and combined, by a deterministic additive model including probability, frequency, and intensity. Furthermore, occupations and industries of longest duration were analysed. Relative risks were estimated by odds ratios (OR), with 95% confidence intervals (CI), using conditional logistic regression, adjusted for potential confounders. RESULTS: Tendencies of positive associations were found between high exposure to pesticides and risk of oesophageal (OR 2.3 (95% CI 0.9 to 5.7)) and cardia adenocarcinoma (OR 2.1 (95% CI 1.0 to 4.6)). Among workers highly exposed to particular agents, a tendency of an increased risk of oesophageal squamous cell carcinoma was found. There was a twofold increased risk of oesophageal squamous cell carcinoma among concrete and construction workers (OR 2.2 (95% CI 1.1 to 4.2)) and a nearly fourfold increased risk of cardia adenocarcinoma among workers within the motor vehicle industry (OR 3.9 (95% CI 1.5 to 10.4)). An increased risk of oesophageal squamous cell carcinoma (OR 3.9 (95% CI 1.2 to 12.5)), and a tendency of an increased risk of cardia adenocarcinoma (OR 2.8 (95% CI 0.9 to 8.5)), were identified among hotel and restaurant workers. CONCLUSIONS: Specific airborne occupational exposures do not seem to be of major importance in the aetiology of oesophageal or cardia adenocarcinoma and are unlikely to contribute to the increasing incidence or the male predominance.

Muscarinic receptor-linked elevation of cAMP in SH-SY5Y neuroblastoma cells is mediated by Ca2+ and protein kinase C.

The mechanisms of muscarinic receptor-linked increase in cAMP accumulation in SH-SY5Y human neuroblastoma cells has been investigated. The dose-response relations of carbachol-induced cAMP synthesis and carbachol-induced rise in intracellular free Ca2+ were similar. The stimulated cAMP synthesis was inhibited by about 50% when cells were entrapped with the Ca2+ chelator BAPTA or in the presence of the protein kinase C (PKC) inhibitor staurosporine. Production of cAMP could be induced also by the Ca2+ ionophore, ionomycin and by TPA, an activator of PKC. When added together TPA and ionomycin had a synergistic effect. When cAMP synthesis was activated with cholera toxin, PGE1 or PGE1 + pertussis toxin carbachol stimulated cAMP production to the same extent as in control cells. Ca2+ and protein kinase C thus seem to be the mediators of muscarinic-receptor linked cAMP synthesis by a direct action on adenylate cyclase.

Effects of the phospholipid environment in the plasma membrane on receptor interaction with the adenylyl cyclase complex of intact cells.

In this study we have examined the effects of variations of the plasma membrane phospholipid and cholesterol content on the metabolic functions of the adenylyl cyclase complex in intact cells. Exposure of cells to 0.1 U/ml of sphingomyelinase led to the degradation of 75, 55 and 40% of the cellular total sphingomyelin mass in human skin fibroblasts (HSF), Chinese hamster lung fibroblasts (CHLF) and rat liver hepatocytes (RLH), respectively. Degradation of sphingomyelin in native cells led in turn to a reduction (within 60 min) of the plasma membrane cholesterol content (by 25, 15 and 10%, respectively). This manipulation of the plasma membrane lipid content did not affect the forskolin or prostaglandin E1-induced activation of adenylyl cyclase (as measured from the conversion of [3H]adenine via [3H]ATP to [3H]cAMP). These manipulations did, however, increase the basal rate of [3H]cAMP formation in rat liver hepatocytes (but not in the fibroblast cell types). With Chinese hamster lung fibroblasts, transfected to express an alpha 2-adrenergic receptor, it was observed that the alpha 2-adrenergic receptor-induced inhibition of adenylyl cyclase activity was slightly (but significantly) diminished in sphingomyelin and cholesterol-depleted cells. With isolated rat liver hepatocytes it was observed that the glucagon (receptor) mediated activation of adenylyl cyclase was also reduced in sphingomyelinase-treated cells. In another set of experiments, CHLF and RLH cells were exposed for 2 h to vesicles prepared from dilauroylphosphatidylcholine, to increase the lateral packing density in the outer leaflet of the plasma membrane. In such treated cells, the receptor-coupling to adenylyl cyclase was markedly reduced both in CHLF (the alpha 2-adrenergic receptor) and RLH (the glucagon-receptor) cells. We conclude that the direct activation of adenylyl cyclase (i.e., by forskolin) is not markedly affected by manipulations outer leaflet phospholipid composition (either reduction of sphingomyelin or increase of phosphatidylcholine), whereas receptor-coupled events clearly are.

Rapid turn-over of plasma membrane sphingomyelin and cholesterol in baby hamster kidney cells after exposure to sphingomyelinase.

Plasma membrane sphingomyelin in baby hamster kidney (BHK-21) cells was hydrolyzed with sphingomyelinase (Staphylococcus aureus) and the effects on membrane cholesterol translocation and the properties of membrane bound adenylate cyclase and Na+/K(+)-ATPase were determined. Exposure of confluent BHK-21 cells to 0.1 U/ml of sphingomyelinase led to the degradation (at 37 degrees C) of about 60% of cell sphingomyelin. No simultaneous hydrolysis of phosphatidylcholine occurred. The hydrolysis of sphingomyelin subsequently led to the translocation (within 40 min) of about 50-60% of cell [3H]cholesterol from a cholesterol oxidase susceptible pool to an oxidase resistant compartment. The translocation of [3H]cholesterol from the cell surface to intracellular membranes was accompanied by a paralleled increase in [3H]cholesterol ester formation. When cells were first exposed to sphingomyelinase (to degrade sphingomyelin) and then incubated without the enzyme in serum-free media, the mass of cell sphingomyelin decreased initially (by 60%), but then began to increase and reached control levels within 3-4 h. The rapid re-synthesis of sphingomyelin was accompanied by an equally rapid normalization of cell [3H]cholesterol distribution. The re-formation of cell sphingomyelin also led to a decreased content of cellular [3H]cholesterol esters, indicating that unesterified [3H]cholesterol was pulled out of the cholesterol ester cycle and transported to the cell surface. Exposure of BHK-21 cells to sphingomyelinase further led to a dramatically decreased activity of ouabain-sensitive Na+/K(+)-ATPase, whereas forskolin-stimulated adenylate cyclase activity was not affected. The activity of Na+/K(+)-ATPase returned to normal in parallel with the normalization of cell sphingomyelin mass and cholesterol distribution. We conclude that sphingomyelin has profound effects on the steady-state distribution of cell cholesterol, and that manipulations of cell sphingomyelin levels directly and reversibly affects the apparent distribution of cholesterol. Changes in the lipid composition of the plasma membrane also appears to selectively affect important metabolic reactions in that compartment.

Functional analysis of the human alpha 2C-C4 adrenergic receptor in insect cells expressed by a luciferase-based baculovirus vector.

A click beetle luciferase-based baculovirus expression vector is described for functional analysis and high level expression of a human alpha 2-adrenergic receptor (alpha 2AR) in Sf9 insect cells. The resultant recombinant baculovirus construct, AcLucGR-alpha 2(C4), was isolated by utilizing the light emitting properties of luciferase and used for abundant expression of the alpha 2C-C4 receptor protein in this lepidopteran insect cell line. A maximal expression of alpha 2-receptors at a level of 1.370 pmol/mg protein was obtained at 48 h after infection as determined by ligand-binding experiments using the alpha 2-receptor antagonist, [3H]rauwolscine. The receptor agonists, noradrenaline and clonidine, displaced the [3H]rauwolscine binding with Ki values 12.3 +/- 1.54 microM and 1.23 +/- 0.11 microM, respectively. The recombinant receptors were functionally intact since the agonists inhibited forskolin-stimulated cAMP production. Here, however, the maximal inhibition was obtained at 36 h after the infection. The results presented here, suggest that the baculovirus expression vector system (BEVS) provides a simple method for abundant expression of functional alpha 2-receptor subtypes. In addition, co-expression of luciferase proved to be useful for screening and isolation of the recombinant baculovirus.

Stable expression of recombinant human alpha 2-adrenoceptor subtypes in two mammalian cell lines: characterization with [3H]rauwolscine binding, inhibition of adenylate cyclase and RNase protection assay.

Cloning of the genes encoding distinct subtypes of human alpha 2-adrenergic receptors (alpha 2-AR) allows the separate recombinant expression of each individual subtype in heterologous systems. We report here the transfection, selection and preliminary pharmacological characterization of two mammalian cell lines, adherent Shionogi S115 mouse mammary tumour cells and human B-lymphoblastoid IBW4 cells growing in suspension, expressing the human alpha 2-AR subtypes alpha 2-C4 and alpha 2-C10 at densities of approx. 2 x 10(5) receptors/cell. Transfection of the subtype genes was verified using a specific RNase protection assay. Pharmacological characterization was carried out with [3H]rauwolscine binding, which was inhibited by oxymetazoline and prazosin in a subtype-selective manner. The sensitivity of (-)-noradrenaline binding to the GTP-analogue 5'-guanylylimidodiphosphate suggested that the receptors are coupled to G-proteins. This was verified in S115 cells by efficient inhibition of forskolin-stimulated cAMP production by the alpha 2-AR agonists, (-)-noradrenaline and clonidine. These cell lines thus appear to be suitable for pharmacological studies on receptor function and ligand binding.

Analysis of Borrelia burgdorferi IgG antibodies with a combination of IgG ELISA and VlsE C6 peptide ELISA.

Enzyme-linked immunosorbent assays (ELISAs) were used to detect antibodies to the C6 peptide of the Borrelia burgdorferi VlsE protein and a selection of B. burgdorferi IgG antigens, separately and as a combination, in 355 serum specimens from blood donors and patients. Western immunoblotting was used as the reference method. The sensitivity of the combined analysis of IgG antigen and C6 peptide analysis was markedly superior to those of the separate analyses. When the C6 peptide and IgG results were concordant, the customary confirmatory Western immunoblotting assay could be omitted, thus reducing the time and cost of analysis.

Growth response to growth hormone (GH) treatment relates to serum insulin-like growth factor I (IGF-I) and IGF-binding protein-3 in short children with various GH secretion capacities. Swedish Study Group for Growth Hormone Treatment.

The purpose of the study was to evaluate the relationship between the 1-yr (n = 193) and 2-yr (n = 128) growth response and the individual serum concentrations of insulin-like growth factor I (IGF-I) and IGF-binding protein 3 (IGFBP-3) before and during GH treatment. Our study group of prepubertal short children had from very low to high GH secretory capacity, estimated during an arginine-insulin tolerance test, and the ages ranged from 3-15 yr at the start of treatment. Their serum levels of IGF-I and IGFBP-3 were low before treatment compared to those in an age-related reference group of prepubertal children and increased significantly from the start to 1 month of GH treatment. The mean increase in height SD score was 0.80 SD score after 1 yr of GH treatment and 1.26 SD score after 2 yr, with a wide range. In univariate analyses the highest correlation coefficients to the 2-yr growth response were found to be vs. the following variables from the start of treatment: IGF-I SD score (r = -0.49), log maximum GH concentration (log GHmax) during the arginine-insulin tolerance test (r = -0.47), difference between the height SD score of the individual child and the midparental height SD score (diffSD score; r = -0.45), IGFBP-3 SD score (r = -0.39), age (r = -0.30), short term change in IGFBP-3 SD score (r = 0.37), and IGF-I SD score (r = 0.34). In multivariate stepwise regression analysis, 41% of the variation in the 2-yr growth response could be explained by IGF-I SD score or log GHmax together with age at the start of treatment, weight SD score at 1 yr of age, and diffSD score. When both IGF-I SD score and GHmax were included and when the short term changes in IGF-I SD score were added, 46% and 58% of the variation, respectively, could be explained. The regression algorithms using different combinations of variables and their corresponding prediction intervals are also presented.

Changes in serum insulin-like growth factor I (IGF-I) and IGF-binding protein-3 levels during growth hormone treatment in prepubertal short children born small for gestational age.

The aims of this study were to describe the basal serum levels of insulin-like growth factor I (IGF-I) and IGF-binding protein-3 (IGFBP-3) and to evaluate their changes during daily GH treatment (0.1 IU/kg; 33 micrograms/kg) for up to 2 yr in prepubertal short children born small for gestational age (SGA) and to correlate these changes with the growth response to GH therapy. Seventy-two prepubertal short children (height SD score, -5.4 to -2.0; age, 2.0-12.9 yr) born SGA (54 boys and 18 girls), eight of whom (six boys and two girls) had signs of Silver-Russell syndrome, participated in the study. The serum concentrations of IGF-I and IGFBP-3 were converted into SDS using our reference values for prepubertal healthy children. The mean (+/-SD) change in height SDS during the year before the start of GH treatment was 0.1 (0.2) and increased to 0.8 (0.4) during the first year (P < 0.001) and to 0.6 (0.3) during the second year of therapy (P < 0.001). Basal levels of both IGF-I and IGFBP-3 were significantly reduced compared with the reference values. The mean (+/-SD) basal serum concentration of IGF-I was -0.6 (1.1) SD score, and 80% of the SGA children had IGF-I levels below the 50th percentile of the reference group. The corresponding values for IGFBP-3 were -0.4 (1.0) SD score and 63%. The mean IGF-I level increased significantly by 55% from baseline to day 10 of treatment, by 76% on day 90, by 90% after 1 yr, and by 123% after 2 yr of GH treatment. The mean increases in IGFBP-3 were not as great: 25%, 27%, 35%, and 43%, respectively. The 1-yr growth response, expressed as the change in height SD score, correlated negatively with both the basal serum concentration of IGF-I (r = -0.37; P < 0.001) and IGFBP-3 (r = -0.35; P < 0.01), whereas a positive correlation was found to the 10-day percent increase in IGF-I (r = 0.32; P < 0.05). No correlations were found with the initial changes in IGFBP-3. Using a multiple stepwise linear regression analysis, the model using chronological age at the start of GH therapy, the mother's height expressed as a SD score, and the short term percent increase in IGF-I accounted for 42% of the variance in the 1-yr growth response. With the inclusion of 24-h GH profiles, 59% of the variability of the growth response could be explained. It is concluded that short prepubertal children born SGA show an increased growth rate in response to GH therapy. Their mean IGF-I and IGFBP-3 levels before treatment were low and correlated negatively with the growth response to treatment, indicating GH insufficiency. Finally, up to 59% of the variability in the 1-yr growth response to GH treatment could be explained by models using auxological and biochemical variables.

Increased proportion of circulating non-22-kilodalton growth hormone isoforms in short children: a possible mechanism for growth failure.

Current knowledge about the interaction between GH and its receptor suggests that the molecular heterogeneity of circulating GH may have important implications for growth. The aim of this study was to investigate the proportion of circulating non-22-kDa GH isoforms in prepubertal children with short stature (height less than -2 SD score) of different etiologies. We have also evaluated the relationships among the ratio of non-22-kDa GH isoforms, auxology, and spontaneous GH secretion. The study groups consisted of 17 girls with Turner's syndrome (TS), aged 3-13 yr, 25 children born small for gestational age (SGA) without postnatal catch-up growth, aged 3-13 yr; and 24 children with idiopathic short stature (ISS), aged 4-15 yr. The results were compared with those from 23 prepubertal healthy children of normal stature (height +/- 2 SD score), aged 4-13 yr. Serum non-22-kDa GH levels, expressed as a percentage of the total GH concentration, were determined by the 22-kDa GH exclusion assay, which is based on immunomagnetic extraction of monomeric and dimeric 22-kDa GH from serum and quantitation of non-22-kDa GH using a polyclonal antibody-based GH assay. All samples were selected from spontaneous GH peaks in 24-h GH profiles. The median proportion of non-22-kDa GH isoforms was increased in children born SGA (9.8%; P = 0.05) and girls with TS (9.9%; P = 0.01), but not in the group of children with ISS (8.9%), compared with that in normal children (8.1%). Individually, increased proportions of non-22-kDa GH isoforms, with values more than 2 SD above the mean for the normal group, were observed in 5 girls with TS, 5 children born SGA, and 4 children with ISS. In children born SGA, the proportion of non-22-kDa GH isoforms was directly correlated with different estimates of spontaneous GH secretion [mean 24-h GH concentration (r = 0.41; P = 0.04), area under the curve over baseline (r = 0.41; P = 0.04), and GH peak area (r = 0.61; P = 0.003)], whereas it was inversely correlated with height SD score (r = -0.42; P = 0.04). In conclusion, an increased proportion of circulating non-22-kDa GH isoforms was observed at spontaneous GH peaks in some non-GH-deficient short children. Our results suggest that the ratio of non-22-kDa GH isoforms in the circulation may have important implications for normal and abnormal growth.

A new dhfrVIII trimethoprim-resistance gene, flanked by IS26, whose product is remote from other dihydrofolate reductases in parsimony analysis.

A new plasmid-borne gene, dhfrVIII, encoding high-level trimethoprim resistance (TpR) was found in an intestinal Escherichia coli. It seems to be a widely occurring mediator of TpR. Among 973 examined TpR E. coli, the new resistance gene was found in 13 (1.3%) isolates from Sweden, Finland and Nigeria. The new gene was sequenced and found to code for a dihydrofolate reductase (DHFR) of 169 amino acids (M(r) 19005). The dhfrVIII gene on the studied plasmid pLMO226 was observed to be flanked by IS26 elements. The dhfrVIII gene and a 3' unidentified open reading frame (ORF) seem to be borne on a compound transposon with IS26 at its ends, since the configuration of two IS26 flanking dhfrVIII and the unidentified ORF was conserved among the isolates that were probe-positive for the gene. Phylogeny parsimony analysis showed the dhfrVIII-encoded enzyme to be only remotely related to other known plasmid-mediated, drug-resistant DHFR. Only a few of the latter form well-supported monophyletic groups.

Engineering of N-terminal threonines in the D1 protein impairs photosystem II energy transfer in Synechocystis 6803.

Mutants of the cyanobacterium Synechocystis sp. PCC 6803 with N-terminal changes in the photosystem (PSII) II D1 protein were analysed by flash-induced oxygen evolution, chlorophyll a fluorescence decay kinetics and 77 K fluorescence emission spectra. The data presented here show that mutations of the Thr-2, Thr-3 and Thr-4 in D1 do not influence the oxygen evolution. A perturbation on the acceptor side was observed and the importance of the N-terminal threonines for an efficient energy transfer between the phycobilisome and PSII and for stability of the PSII complex was demonstrated.

D1' centers are less efficient than normal photosystem II centers.

One prominent difference between the photosystem II (PSII) reaction center protein D1' in Synechocystis 6803 and normal D1 is the replacement of Phe-186 in D1 with leucine in D1'. Mutants of Synechocystis 6803 producing only D1', or containing engineered D1 proteins with Phe-186 substitutions, were analyzed by 77 K fluorescence emission spectra, chlorophyll a fluorescence induction yield and decay kinetics, and flash-induced oxygen evolution. Compared to D1-containing PSII centers, D1' centers exhibited a 50% reduction in variable chlorophyll a fluorescence yield, while the flash-induced O(2) evolution pattern was unaffected. In the F186 mutants, both the P680(+)/Q(A)(-) recombination and O(2) oscillation pattern were noticeably perturbed.

Agonist trafficking of G(i/o)-mediated alpha(2A)-adrenoceptor responses in HEL 92.1.7 cells.

1. The ability of 19 agonists to elevate Ca(2+) and inhibit forskolin-induced cyclic AMP elevation through alpha(2A)-adrenoceptors in HEL 92.1.7 cells was investigated. Ligands of catecholamine-like- (five), imidazoline- (nine) and non-catecholamine-non-imidazoline-type (five) were included. 2. The relative maximum responses were similar in both assays. Five ligands were full or nearly full agonists, six produced 20 - 70% of the response to a full agonist and the remaining eight gave lower responses (< 20%) so that their potencies were difficult to evaluate. 3. Marked differences in the potencies of the agonists with respect to the two measured responses were seen. The catecholamines were several times less potent in decreasing cyclic AMP than in increasing Ca(2+), whereas the other, both imidazoline and ox-/thiazoloazepine ligands, were several times more potent with respect to the former than the latter response. For instance, UK14,304 was more potent than adrenaline with respect to the cyclic AMP response but less potent than adrenaline with respect to the Ca(2+) response. 4. All the responses were sensitive to pertussis toxin-pretreatment. Also the possible role of PLA(2), beta-adrenoceptors or ligand transport or metabolism as a source of error could be excluded. The results suggest that the active receptor states produced by catecholamines and the other agonists are markedly different and therefore have different abilities to activate different signalling pathways.

Circulating non-22 kDa growth hormone isoforms in healthy children of normal stature: relation to height, body mass and pubertal development.

The proportion of non-22 kDa GH isoforms was evaluated in 93 healthy children (48 boys aged 6.8-18.4 years and 45 girls aged 3.9-18.4 years) of normal stature (height +/- 2 s.d. score) at different stages of puberty. In addition, correlations among the proportion of non-22 kDa GH isoforms, auxology, spontaneous GH secretion and biochemical measurements were investigated. Serum non-22 kDa GH levels, expressed as percentage of total GH concentration in the samples, were determined by the 22 kDa GH exclusion assay, in which monomeric and dimeric 22 kDa GH are removed from serum and the non-22 kDa GH isoforms are quantitated using a polyclonal antibody GH assay. Samples were selected from spontaneous GH peaks in 24-h GH profiles. For boys, the median proportion of non-22 kDa GH isoforms was 8.5% (range 3.2-26.6%) and for girls it was 9.6% (1.8-17.4%), with no influence of age and no sex-related difference in prepubertal (boys, 7.2%; girls, 8.8%) or pubertal children (boys, 9.1%; girls, 9.9%). However, the median proportion of non-22 kDa GH isoforms was significantly higher in pubertal boys (9.1%) than in prepubertal boys (7.2%; P = 0.03). In pubertal boys, height S.D. scores (SDS) were inversely correlated to the proportion of non-22 kDa GH isoforms (r = -0.38; P = 0.02), especially at mid-puberty (r = -0.7; P = 0.01), indicating that the presence of increased amounts of circulating non-22 kDa GH isoforms was associated with less growth. In prepubertal children, positive correlations between non-22 kDa GH and weight SDS (r = 0.46; P = 0.03), weight-for-height SDS (r = 0.51; P = 0.01) and body mass index (r = 0.42; P = 0.04) were observed. No significant correlations were seen with spontaneous GH secretion or measurements of IGF-1, IGF-binding protein-3, insulin and leptin. These findings in normal children indicate that the proportion of circulating non-22 kDa GH isoforms may have physiologic significance for growth and metabolism in different stages of development, and emphasize the importance of evaluating the circulating ratio of 22 kDa and non-22 kDa GH in children with growth disorders.

Transformation of nuclear and plastomic plant genomes by biolistic particle bombardment.

Microprojectile bombardment is a powerful method for the transformation of various organisms and tissues. For plants, the biolistic approach is primarily used for transformation of cereals and other monocotyledons, as well as for dicotyledonous plants shown to be recalcitrant to Agrobacterium-based transformation of organellar genomes, and transformation of plant and algal chloroplasts has recently been reported. In this protocol paper we provide methods for nuclear and plastomic transformation of plants using the biolistic technique.

Differential coupling of muscarinic receptors to Ca2+ mobilization and cyclic AMP in SH-SY5Y and IMR 32 neuroblastoma cells.

Muscarinic receptor-linked Ca2+ mobilization and changes in cyclic AMP were studied in SH-SY5Y and IMR 32 human neuroblastoma cell lines. Muscarinic agonists acetylcholine, carbachol, methacholine and muscarine induced an increase in cytosolic free Ca2+ in a pertussis toxin (100 ng/ml)-insensitive manner in both cell lines. The ED50 values in IMR 32 cells (8-98 microM) were one order of magnitude higher than in SH-SY5Y cells (0.3-1.6 microM). Oxotremorine and pilocarpine failed to mobilize Ca2+ in IMR 32 cells. Pirenzepine antagonized carbachol-induced Ca2+ mobilization in SH-SY5Y cells with a Ki value in the range of 150-189 nM whereas the corresponding values in IMR 32 cells were 24-28 nM. Atropine inhibited a carbachol-stimulated increase in cytosolic Ca2+ with an equal potency in both cell lines (Ki 2-3 nM). Carbachol stimulated cyclic AMP (cAMP) accumulation in SH-SY5Y cells in a pertussis toxin-insensitive manner. In IMR 32 cells carbachol inhibited prostaglandin E1-stimulated cAMP accumulation. Treatment of IMR 32 cells with pertussis toxin abolished the inhibition of stimulated cAMP accumulation. These results suggest that in SH-SY5Y cells the M3 muscarinic receptor couples to both Ca2+ mobilization and stimulation of cAMP accumulation. In IMR 32 cells the M1 receptor seems to couple to Ca2+ mobilization whereas the inhibition of stimulated cAMP accumulation is coupled to a non-M1 subtype by an inhibitory G-protein.

Assessment of alpha2-adrenoceptor antagonist potency with GTPase assay.

Membranes from Chinese hamster ovary (CHO) cells expressing high densities of alpha2A-, alpha2B- or alpha2C-adrenoceptor subtypes were used to monitor potencies of alpha2-adrenoceptor antagonists with a GTPase assay. Receptor-activated high-affinity GTPase activity was determined by measuring the rate of release of 32P from [gamma-32P]GTP. Concentration-response curves to the full agonist (-)-noradrenaline were obtained in the presence of different antagonist concentrations and pA2 values were calculated by Schild analysis. Three antagonists (rauwolscine, prazosin and chlorpromazine) showed subtype-selectivity, which agrees with earlier radioligand binding results. We suggest that the GTPase assay described here is useful for characterization of the functional potency and subtype-selectivity of alpha2-adrenoceptor antagonists.