Identification and molecular mapping of indica high-tillering dwarf mutant htd4, a mild phenotype allelic mutant of D14 in rice (Oryza sativa L.).

Metabolism of strigolactones (SLs) can improve the efficiency of nutrient use by regulating the development of roots and shoots in crops, making them an important research focus for molecular breeding. However, as a very important plant hormone, the molecular mechanism of SL signal transduction still remains largely unknown. In this study, we isolated an indica high-tillering dwarf mutant 4 (htd4), a spontaneous mutant of rice, from the restorer line Gui99. Mapping and sequencing analysis showed that htd4 was a novel allelic mutant of D14, in which a single base substitution forms a premature termination codon. Quantitative RT-PCR analyses revealed that expression levels of the genes D10, D17, D27, D3 and D14 increased significantly, while expression of D53 decreased in htd4, compared with the wild type. A subcellular localisation assay showed that the mutant of D14 in htd4 did not disturb the normal localisation of D14 proteins. However, a BiFC assay suggested that the mutant-type D14 could not interact with D3. Additionally, compared with other D14 allelic mutants, htd4 was the first mutant of D14 discovered in indica, and the differences in many yield traits such as plant height, seed-setting rate and grain sizes between htd4 and the wild type were less than those between other D14 allelic mutants and the wild type. Therefore, htd4 is considered a mild phenotype allelic mutant of D14. We conclude that the absence of functional D14 caused the high-tillering dwarf phenotype of htd4. Our results may provide vital information for research on D14 function and the application of htd4 in molecular breeding.

Effect of insulin on oogenesis from mouse fetal germ cells in a serum-free 3D culture system.

Continuous exposure of oocytes to elevated concentrations of insulin compromises embryonic developmental competence. However, the effects of insulin on oogenesis from fetal germ cells are unknown. The objective of this study was to assess the effect of continuous insulin exposure, with or without FSH, on oogenesis and follicular development. A simple and efficient method was established that could be used to obtain oocytes from pre-meiotic germ cells in 12.5days post-coitum (dpc) fetal mouse ovaries using a three-dimensional culture system with serum-free medium. Mouse 12.5dpc fetal ovaries were cultured for 14days with or without insulin/FSH. Low (0.2-1microg/ml) or high (5-20microg/ml) doses of insulin retarded oocyte growth in vitro. Insulin at 5microg/ml led to significant oocyte growth retardation (P<0.05), while FSH alleviated the deleterious effect of insulin. Most importantly, the proportion of secondary follicles at 12days post-culture in the presence of insulin was reduced significantly compared with controls (P<0.05). Expression levels of genes specific for ovarian cells, e.g. Cx37, Cx43, Scp3, Bax and FSHR, were significantly reduced when exposed to insulin during oogenesis (P<0.05). The data suggest that insulin has a profound detrimental effect on oogenesis and folliculogenesis in vitro.

Premeiotic fetal murine germ cells cultured in vitro form typical oocyte-like cells but do not progress through meiosis.

A convenient method for fetal murine premeiotic germ cells to develop into oocytes in vitro has been established. Fetal ovaries from mice, collected 12.5 d postcoitus (dpc), were organ-cultured in vitro using a medium for organ growth, and the developmental potential regarding oocyte formation was determined. After 28 d of culture, premeiotic female germ cells developed into oocytes with a mean (+/-SD) diameter of 73.3+/-7.7 microm. However, follicles developed in vitro versus in vivo had fewer granulosa cells (32+/-2.6 vs. 142+/-9.5, respectively; P<0.01), and the ovaries had less mRNA for Cx37 and Cx43 (P<0.01). Oocytes in the first meiotic division phase were isolated from cultured ovaries or after hormone treatments. After exposure to okadaic acid at a final concentration of 1 microM, oocytes derived from premeiotic fetal female germ cells were able to undergo germinal vesicle breakdown but failed to complete the first meiotic division. Furthermore, the intracellular content of GSH in oocytes cultured in vitro was lower than that of oocytes matured in vivo (P<0.01). In conclusion, premeiotic germ cells derived from murine fetuses as early as 12.5 dpc were able to differentiate into germinal vesicle-stage oocytes but were unable to complete meiosis I in vitro.

Recombinant human erythropoietin administration protects cortical neurons from traumatic brain injury in rats.

We explored the regulation of erythropoietin and erythropoietin receptor on traumatic brain injury (TBI), as well as the antiapoptotic effects of recombinant human erythropoietin (rhEPO) treatment. Female Wistar rats were randomly divided into three groups: rhEPO-treated TBI, vehicle-treated TBI, and sham-operated. TBI was induced by the Feeney free falling model. Rats were killed 5, 12, 24, 72, 120, or 168 h after TBI. Regulation of EPO, EPOR and Bcl-2 was detected by reverse transcription-polymerase chain reaction (RT-PCR), western blotting and immunofluorescence. Terminal deoxynucleotidyl transferase-mediated biotin-dUTP nick-end labeling (TUNEL) was used to assess DNA fragmentation after TBI. Induction of EPOR expression persisted for 168 h after TBI, whereas EPO was only slightly elevated for 72 h. In the rhEPO-treated TBI, Bcl-2 mRNA and protein levels were greater than in the vehicle-treated TBI. Bcl-2 mRNA peaked at 24 h and remained stable for 72-120 h. The number of TUNEL-positive cells in the rhEPO-treated TBI was far fewer than in the vehicle-treated TBI. EPOR regulation is enhanced for almost a week after TBI. Administration of rhEPO protects neurons by enhancing Bcl-2 expression, thereby inhibiting TBI-induced neuronal apoptosis.

[Detection of numerical chromosomal aberrations in epididymal sperm of mice using three-color FISH with chromosome-specific DNA probes].

To detect malsegregation of chromosomes during meiosis in male mice, three-color FISH using DNA probes specific for mouse chromosomes X, Y and 8 was performed on epididymal sperm decondensed for 30 min each in 10 mmol/L DTT and 4 mmol/L LIS, and conventional chromosome counting was also carried out on C-banded MMII cells. Comparisons between these two methods indicate as the following: (1) three-color FISH is a simple, rapid and sensitive approach; (2) The estimate of the frequency of aneuploidy obtained from three-color FISH analysis is more reliable since extremely large numbers of sperm (more than 10,000 sperm per animal) are scored; (3) The rates of cells arrested at meiosis I and meiosis II (representing diploid sperm) can be detected only by using the three-color FISH approach; (4) Malsegregation of chromosomes occurred during both meiosis I and meiosis II can be detected simultaneously by using this new approach. The probes and scoring criteria used in FISH on interphase sperm nuclei, and the necessity of using three or more chromosome-specific DNA probes to thoroughly evaluate aneugenic effects of factors tested on meiosis in male mice were discussed.

[Studies of meiotic origin of the extra chromosome 21 in Down syndromes detected by using (GT)n polymorphic DNA markers].

The polymorphics of two pericentric (GT)n sequences on the long arm of human chromosome 21 have been analyzed after PCR amplification, PAGE and Ag-staining for the first time in 50 Chinese Han people, and were used to detect meiotic origin of the extra chromosome 21 in Down syndromes. Six and 5 alleles were found in Chinese Han people for D21S215 and D21S120, respectively, with observed heterozygosities of 0.68 and polymorphic information content PIC, 0.67 and 0.65. For 17 Down syndromes whose parental origin of the extra chromosome 21 were known, meiotic origin of the extra chromosome 21 were determined in 16 cases, with 7 and 4 maternal meiosis I and II nondisjunction, 2 and 3 paternal meiosis I and II, respectively. The possible biological significance of the study on origin of the extra chromosome 21 has been discussed.

[Studies on meiotic delay and aneuploidies induced by colcemid (COM) in male mouse germ cells].

Colcemid (COM) was tested for induction of meiotic delay and aneuploidies in meiotic metaphase II (MMII) of male(101/E 1 XC 3 H/E 1)F 1 mice post single intraperitoneum (i.p.) injection. The dose of 1 mg/kg of COM was used and sampled at 2, 6, 10, 14 and 18 h after COM treatment. The number of MMII and MMI were the highest at 2 and 6 h repectively, and decreased rapidly with lengthening of COM treatment and reached the lowest at 14 h; then increased. However, the ratio of MMII to MMI was always significantly higher than in solvent control at every sample interval. Under our experimental conditions, COM did not show aneugenicity in male mouse germ cells. The possible mechanisms by which COM caused meiotic delay and reasons that COM did not induce aneuploidy in MMII were discussed.

[Spectrophotometric determination of complex formation constants of naproxen with caffeine, nicotinamide and salicylic acid in aqueous solution].

The interaction between water insoluble drug naproxen with caffeine, nicotinamide and salicylic acid in aqueous solutions were determined by spectrophotometry. Naproxen was found to form 1:1 molecular complex with these substances. The complex formation constants were determined by spectrophotometry at room temperature. The thermodynamic functions associated with complexation of naproxen-drugs were also evaluated. It may be concluded that the complex formation may be attributed to hydrogen bonding since enthalpy changes were shown to be a few KJ.mol-1.

A dose-response study of forskolin, stimulatory hormone, and guanosine triphosphate analog on adenylate cyclase from several sources.

We have described relationships involving forskolin stimulation of adenylate cyclase (AC) from a variety of sources and the potentiation of forskolin effects by stimulatory hormones (glucagon, ACTH, and epinephrine) and beta, gamma-imidoguanosine 5'-triphosphate (Gpp(NH)p). The effects on AC were examined using membrane preparations of rabbit adipocytes, rat adipocytes, rat erythrocytes, and rat liver. Also examined was the AC of liver membranes of rat pretreated with pertussis toxin as well as that solubilized from rat liver membranes. Maximal forskolin stimulation of AC in all preparations studied revealed a consistent 10-fold increase in AC activity. The EC50 for forskolin was 10 microM for rat liver, 15 microM for rabbit and rat adipocytes and 17 microM for rat erythrocyte AC stimulation. In all cases the AC activity attained by forskolin stimulation was further enhanced by stimulatory hormones in a dose-dependent manner. Furthermore, a combination of all three activators (forskolin, stimulatory hormone, and Gpp(NH)p) resulted in an even greater overall stimulation to levels ranging from 25- to 30-fold over unstimulated activity levels. In the presence of saturating levels of each stimulatory hormone and Gpp(NH)p, the EC50 for forskolin diminished markedly to the range of 0.5 to 4.0 microM. In the absence of any apparent tissue specificity for forskolin stimulation, the general pattern of these results further implicates the catalytic site of the AC complex as the site of forskolin activation. Furthermore, activation of additional components of the complex by Gpp(NH)p and tissue specific hormones may further influence the AC activity and thereby potentiate the stimulation by forskolin.

Conditional inhibition of forskolin-activated adenylate cyclase by guanosine diphosphate and its analog.

Forskolin-activated adenylate cyclases (AC) in intact membranes, solubilized with Lubrol or eluted following adsorption on a forskolin-Sepharose column, were examined for inhibition by GDP and GDP beta S. AC in intact membranes of rat or rabbit adipocytes was activated by 100 microM forskolin and further potentiated by 10 microM Gpp(NH)p in combination with either 230 microM epinephrine or 50 mU X ml-1 ACTH. GDP (0-1 mM) or GDP beta S (0-500 microM) inhibited activation in a dose-dependent manner to a level similar to or slightly below that produced by 100 microM forskolin alone. Forskolin at 100 microM stimulated solubilized AC of rabbit adipocytes and rat liver membranes for 10 +/- 4 to 160 +/- 10 and from 26 +/- 2 to 274 +/- 21 pmol(mg X min)-1, respectively, in the absence of GDP beta S; forskolin-activated activity decreased from 160 +/- 10 to 157 +/- 6 and from 274 +/- 21 to 238 +/- 14 pmol(mg X min)-1 in the presence of 500 microM GDP beta S. Forskolin-activated solubilized enzyme was further potentiated by 10 microM Gpp(NH)p from 160 +/- 10 to 289 +/- 52 and from 274 +/- 21 to 702 +/- 50 pmol(mg X min)-1. GDP beta S at 500 microM inhibited 93 and 103% of the Gpp(NH)p-potentiated activity. AC of rat adipocytes eluted from forskolin-Sepharose affinity column with 500 mM NaCl and 100 microM forskolin was not significantly activated by Gpp(NH)p nor inhibited by GDP beta S. However, it was activated by forskolin. The lack of inhibition of unmodified forskolin-activated activity by GDP or GDP beta S in contrast to the inhibition of Gpp(NH)p-activated enzyme or Gpp(NH)p-potentiated forskolin-activated enzyme may be a general phenomenon descriptive of the action of forskolin on AC. Furthermore, inhibition of forskolin-activated AC by GDP and its analog may be a useful index in analyzing the degree of guanine nucleotide potentiation of this enzyme.

Forms of adenylate cyclase, activation and/or potentiation by forskolin.

Activation of different forms of adenylate cyclases (AC) by forskolin and displacement of [14,15-3H]dihydroforskolin binding from membranes by forskolin in the absence or presence of specific stimulatory hormone and beta, gamma-imidoguanosine 5'-triphosphate (Gpp(NH)p) have been studied. These conditions have been used to generate forskolin dose-response curves of AC activation. A plot of enzyme activation versus apparent forskolin-binding showed a linear and a nonlinear relationship, respectively, in the absence or presence of the other two stimulators. The latter relationship can be fitted by two linear regression lines with a defined intercept, the slopes of which represent two distinct binding-activation (B-A) effects. The B-A effects of forskolin for rat adipocyte and liver membranes in the absence of stimulatory hormone and Gpp(NH)p were 10 and 8 (pmol X min-1) X (pmol)-1, respectively. The B-A effects for the same membranes in the presence of the other two stimulators were 69 (high) and 13 (low) (pmol X min-1) X (pmol)-1 for adipocyte membrane, and 83 (high) and 9 (low) (pmol X min-1) X (pmol)-1 for liver membrane. The ratio of potentiation of forskolin-activated enzyme activity to the unmodified forskolin-stimulated activity (P-A ratio) was determined without the binding data. At 3 microM forskolin, with and without 230 epinephrine and 10 microM Gpp(NH)p, the P-A ratio was 3.7, decreasing to 1.1 with the addition 100 microM forskolin. The line representing a high B-A effect and a resulting high P-A ratio appears to describe the interactions between forskolin and the AC stimulated by epinephrine and Gpp(NH)p. The line of low B-A effect may represent the interaction between forskolin and the basal AC. Two peaks of AC activity were eluted from forskolin-Sepharose column. They have apparent differences in sensitivity to Gpp(NH)p and affinity for forskolin. Based on the results available thus far, with consideration for known limitations of the methodology, a working model has been proposed for forskolin activation of AC.

Evidence for a single forskolin-binding site in rat adipocyte membrane. Studies of [14,15-3H]dihydroforskolin binding and adenylate cyclase activation.

[14,15-3H] Dihydroforskolin has been used as a tracer in the study of forskolin binding to adipocyte plasma membrane and the subsequent activation of adenylate cyclase (EC 4.6.1.1) of this membrane. The specific binding of radioactivity to the membrane was rapid, temperature-dependent, saturable, and readily reversible. The equilibrium dissociation reaction constant (KD) for the binding was 13 microM, with a maximum binding (Bmax) of 61 pmol of forskolin per mg of membrane protein. The Hill coefficient was 1.0. The bound [14,15-3H] dihydroforskolin was displaced by forskolin with rate constants of 0.07 X 10(6) M-1 min-1 and 1.2 min-1 for the association and dissociation reactions, respectively (30 degrees C). The equilibrium dissociation constant (KD) was approximately the same as the concentration that produced half-maximum activation (EC50) of the adenylate cyclase of rat adipocyte plasma membrane. There was a linear correlation between forskolin binding and adenylate cyclase activation. The results are consistent with the concept of a single class of binding site which binds forskolin. [14,15-3H] Dihydroforskolin appears to be a potentially useful tracer in the study of the mechanism of activation of the catalytic unit of adipocyte adenylate cyclase.

Forskolin as a probe of adenylate cyclase of adipocytes.

New information regarding the relationship between the agonist receptor and the regulatory and catalytic components of adenylate cyclase has been obtained by applying forskolin in combination with several different activators of adenylate cyclase. Adipocyte adenylate cyclase cannot be fully activated by forskolin; maximal stimulation by forskolin can be further enhanced by Gpp(NH)p. The effect of Gpp(NH)p on membrane enzyme differs between rat and rabbit and between enzymes of membrane-bound and solubilized preparations of rat adipocytes. Conditions that produce maximal activation of the membrane adenylate cyclase have been found. Gpp(NH)p and NaF are both activators of adenylate cyclase but mutually oppose each other. These findings indicate multiple activatable forms of catalytic components and multiple activatable forms of regulatory components of adenylate cyclase.

Cyclic AMP-lowering mediator of insulin.

What appears to be a mediator of insulin action has been successfully produced in rat adipocytes plasma membrane upon its treatment with insulin at concentrations of 50-200 microunits/ml. This apparent mediator, when isolated and presented to adipocyte cells, mimics insulin action in the lowering of hormonally stimulated cAMP levels as well as in stimulating lipogenesis and antilipolysis. The cAMP-lowering activity of such a mediator can be quantitated as insulin-activity equivalents. Insulin at 200 microunits/ml causes, in terms of insulin-activity equivalents, a generation of as much as 50 times more of insulin mediator. The magnitude of amplification is even greater when the amount of insulin bound to its receptor is taken into account in this calculation. The action of insulin in the generation of cAMP-lowering mediator is abolished by the insulin antibody. Inactive insulin analogs do not effectively generate such a mediator activity. On the other hand, while the cAMP-lowering action of insulin shown in the bioassay system is completely inhibited by the insulin antibody, the action of such a mediator is only slightly inhibited by the same antibody. The mediator has a low molecular weight and attributes that resemble a peptide. It can be separated from insulin in a Sephadex G-25 column and has a molecular size smaller than the insulin A-chain but larger than ATP. The molecular weight of this mediator is similar to the insulin mediators isolated by other investigators. In view of the fact that it is small in size and mimics several actions of insulin when used in extracellular situations, its theoretical, as well as practical, implications are substantial.