Elaboration and characterization of a 200 mm stretchable and flexible ultra-thin semi-conductor film.

We describe a process for transferring a 200 nm thick, 200 mm wide monocrystalline silicon (mono c-Si) thin film from a silicon-on-insulator onto a flexible polymer substrate. The result is a stretchable and flexible ultra-thin semi-conductor film that can be subjected to tensile stress experiments. The process uses off-the-shelf 200 mm wafers and standard polymer temporary bonding techniques. The backside substrate and buried oxide are removed using grinding and wet etching processes. No cracks or wrinkles are observed on the film prior to the tensile stress experiments. The stretching of the flexible structure results in up to 1.5% uniaxial tensile elastic strain on the thin mono c-Si film.

Crack Front Interaction with Self-Emitted Acoustic Waves.

The interaction of a propagating crack in implanted silicon with self-emitted acoustic waves results in periodic patterns on fractured surfaces. Direct measurement of the acoustic emission ahead of the fracture front shows the emergence of dominant acoustic frequency related to the crack velocity. It is shown that the surface modifications are made of roughness modulations due to periodic deviations of the crack front. A physical mechanism explaining the pattern formation is proposed, well in agreement with the observed pattern wavelengths.

Developmental plasticity of the microscopic placental architecture in relation to litter size variation in the common marmoset monkey (Callithrix jacchus).

Fetal demand, shaped by factors such as number of fetuses, may alter placental regulation of exchange, even when maternal nutrition restriction is not overt. The marmoset is an interesting model in which to examine this aspect of placental function due to unique placentation that leads to multiple fetuses sharing a unified placental mass. We demonstrated previously that the triplet marmoset placenta exhibits significantly higher efficiency than does the twin placenta. Here, we test the hypothesis that this increased efficiency is due to increases in changes in the microscopic morphology of the placenta. Stereology was employed to analyze the microscopic architecture of placentas from twin and triplet pregnancies. Compartments of interest were the trabeculae, intertrabecular space, fetal capillaries, and the surface area of the maternal-fetal interface. Placentas from the two litters did not differ significantly in overall volume or individual volumetric compartments, but triplet placentas exhibited significant expansion of the trabecular surface area in comparison to twins (p=0.039). Further, the two groups differed in the isomorphy coefficient, with triplet placentas having a significantly higher coefficient (p=0.001) and potentially a more complex microscopic topography. Differences in the maternal-fetal interface may be due to developmental constraints on gross placental growth that occur earlier in gestation, such that the only option for maintaining sufficient access to maternal resources or signaling pathways late in gestation is via an expansion of the interface. Despite the significant increase in overall surface area, individual triplet fetuses are associated with much less surface area than are individual twins, suggestive of alterations in metabolic efficiency, perhaps via differential amino acid transport regulation.

Linking bioavailability and toxicity changes of complex chemicals mixture to support decision making for remediation endpoint of contaminated soils.

A six-month laboratory scale study was carried out to investigate the effect of biochar and compost amendments on complex chemical mixtures of tar, heavy metals and metalloids in two genuine contaminated soils. An integrated approach, where organic and inorganic contaminants bioavailability and distribution changes, along with a range of microbiological indicators and ecotoxicological bioassays, was used to provide multiple lines of evidence to support the risk characterisation and assess the remediation end-point. Both compost and biochar amendment (p = 0.005) as well as incubation time (p = 0.001) significantly affected the total and bioavailable concentrations of the total petroleum hydrocarbons (TPH) in the two soils. Specifically, TPH concentration decreased by 46% and 30% in Soil 1 and Soil 2 amended with compost. These decreases were accompanied by a reduction of 78% (Soil 1) and 6% (Soil 2) of the bioavailable hydrocarbons and the most significant decrease was observed for the medium to long chain aliphatic compounds (EC16-35) and medium molecular weight aromatic compounds (EC16-21). Compost amendment enhanced the degradation of both the aliphatic and aromatic fractions in the two soils, while biochar contributed to lock the hydrocarbons in the contaminated soils. Neither compost nor biochar affected the distribution and behaviour of the heavy metals (HM) and metalloids in the different soil phases, suggesting that the co-presence of heavy metals and metalloids posed a low risk. Strong negative correlations were observed between the bioavailable hydrocarbon fractions and the ecotoxicological assays suggesting that when bioavailable concentrations decreased, the toxicity also decreased. This study showed that adopting a combined diagnostic approach can significantly help to identify optimal remediation strategies and contribute to change the over-conservative nature of the current risk assessments thus reducing the costs associated with remediation endpoint.

Polymorphic microsatellite loci for the common marmoset (Callithrix jacchus) designed using a cost- and time-efficient method.

We describe a cost- and time-efficient method for designing new microsatellite markers in any species with substantial genomic DNA sequence data available. Using this technique, we report 14 new polymorphic dinucleotide microsatellite loci isolated from the common marmoset. The relative yield of new polymorphisms was higher with less labor than described in previous marmoset studies. Of 20 loci initially evaluated, 14 were polymorphic and amplified reliably (70% success rate). The number of alleles ranged from 3 to 9 with heterozygosity varying from 0.48 to 0.83.

Insights into mixed contaminants interactions and its implication for heavy metals and metalloids mobility, bioavailability and risk assessment.

Mobility of heavy metals at contaminated sites is mainly influenced by the soil physicochemical properties and environmental conditions, therefore assessing heavy metals (HMs) and metalloids fractionation can provide insights into their potential risk and the mechanisms that regulate bioavailability. A 12-months mesocosms experiment was setup to investigate the effect of physicochemical factors (pH, moisture, and temperature) and weathering (time) on HMs and metalloids fractionation in three different multi-contaminated soil matrices (low, medium, and high contamination) collected from a soil treatment facility located in the United Kingdom, and two rural contaminated soil samples. The study demonstrates that even though Pb and Zn were found associated with the exchangeable fraction in the soil with the highest contamination (total average Pb 3400 mg/kg, and total average Zn 2100 mg/kg in Soil C), neither the condition applied nor the weathering caused an increase in their mobility. Although it was expected that lower pH (4.5) would favours the dissociation of HMs and metalloids, no significant differences were observed, potentially due to the initial alkaline pH of the genuine-contaminated soil samples. The results show that even though total concentration of Pb, Cu, and Zn exceed the soil standards and guideline values, HMs were predominantly associated with the non-exchangeable fraction, while only 5% were dissolved in the pore water fraction (potentially bioavailable). In addition, the mobility and bioavailability of HMs remained constant over the 12 months monitoring, suggesting that these soils pose negligible risk to the environment.

How efficacious are 5-HT1B/D receptor ligands: an answer from GTP gamma S binding studies with stably transfected C6-glial cell lines.

The intrinsic activity of a series of 5-hydroxytryptamine (serotonin, 5-HT) receptor ligands was analysed at recombinant h5-HT1B and h5-HT1D receptor sites using a [35S]GTP gamma S binding assay and membrane preparations of stably transfected C6-glial cell lines. Compounds either stimulated or inhibited [35S]GTP gamma S binding to a membrane preparation containing either h5-HT1B or h5-HT1D receptors. The potencies observed for most of the compounds at the h5-HT1B receptor subtype correlated with their potencies measured by inhibition of stimulated cAMP formation on intact cells. Apparent agonist potencies in the [35S]GTP gamma S binding assay to C6-glial/h5-HT1D membranes were, with the exception of 2-[5-[3-(4-methylsulphonylamino)benzyl-1 2,4-oxadiazol-5-yl]-1H-indol-3-yl] ethanamine (L694247), 5- to 13-times lower than in the cAMP assay on intact cells. This suggests that receptor coupling in the h5-HT1D membrane preparation is less efficient than that in the intact cell. It further appeared that 6-times more h5-HT1D than h5-HT1B binding sites were required to attain a similar, maximal (73%), 5-HT-stimulated [35S]GTP gamma S binding response: Hence, the h5-HT1B receptor in C6-glial cell membranes could be more efficiently coupled, even though some compounds more readily displayed intrinsic activity at h5-HT1D receptor sites [e.g. dihydroergotamine and (2'-methyl-4'-(5-methyl[1,2,4]oxadiazol-3-yl)biphenyl-4-carboxylic acid [4-methoxy-3-(4-methylpiperazin-1-yl)phenyl]amide (GR127935)]. Efficacy differences were apparent for most of the compounds (sumatriptan, zolmitriptan, rizatriptan, N-methyl-3-[pyrrolidin-2(R)-ylmethyl]-1H-indol-5-ylmethyl sulfonamide (CP122638), dihydroergotamine, naratriptan and GR127935) that stimulated [35S]GTP gamma S binding compared to the native agonist 5-HT. The observed maximal responses were different for the h5-HT1B and h5-HT1D receptor subtypes. Few compounds behaved as full agonists: L694247, zolmitriptan and sumatriptan did so at the h5-HT1B receptor and only L694247 at the h5-HT1D receptor. GR127935 (10 microM) exerted little effect on [35S]GTP gamma S binding via h5-HT1B receptors (10% stimulation), but potently (pA2: 9.11) antagonized h5-HT1B receptor-stimulated [35S]GTP gamma S binding. Ketanserin and methiothepin inhibited [35S]GTP gamma S binding (by 13-28%) in the absence of an agonist, but were potent and competitive antagonists in the presence of an agonist via h5-HT1B (methiothepin) and h5-HT1D (methiothepin and ketanserin) receptors. The results document the utility of using [35S]GTP gamma S binding studies to assess agonist efficacy, and to characterize 5-HT1B/D receptor ligands as apparently neutral antagonists and inverse agonists at the G-protein level.

Modulation of 5-HT1A receptor signalling by point-mutation of cysteine351 in the rat Galpha(o) protein.

The activity state of G proteins is involved in the ligands' maximal responses that can be produced by activating the 5-HT1A receptor (Pauwels et al., 1997). The present study investigated the ligand responses at the recombinant h 5-HT1A receptor (RC: 2.1.5HT.01A) as mediated by the Galpha(o) protein. Therefore, a fusion protein was constructed between the 5-HT1A receptor and a pertussis toxin resistant rat Galpha(o)Cys351Gly mutant protein to define its pharmacological properties at a receptor: Galpha(o) protein density ratio of 1. Pertussis toxin treatment (100 ng/ml) affected neither the expression of the 5-HT1A receptor fusion protein as measured by [3H] MPPF (3.0+/-0.7 pmol/mg protein) nor the 5-HT-mediated [35S]GTPgammaS binding response (146+/-34 fmol/mg protein) in Cos-7 cells. 8-OH-DPAT (Emax: 55+/-7%) and buspirone (Emax: 22+/-4%) yielded partial agonist activity as compared to 5-HT, whereas WAY 100635 acted as a competitive antagonist (pK(B): 9.75+/-0.17). The magnitude of the 8-OH-DPAT response (Emax, %) was highly dependent on the nature of the amino acid 351 in the C-terminus of the Galpha(o) protein: Ile351 (93+/-4) > Cys351 (79+/-3) > Gly351 (55+/-7). The Emax values (%) of buspirone displayed the following gradient: 69+/-5 approximately/= 62+/-8 > 22+/-4. For comparison, maximal responses of 8-OH-DPAT and buspirone were enhanced versus 5-HT upon co-expression of the 5-HT1A receptor with the respective Galpha(o) proteins, probably due to an altered receptor: Galpha(o) protein density ratio. In conclusion, residue 351 of the rat Galpha(o) protein is involved in determining the magnitude of 5-HT1A receptor activation that ligands can produce at these receptors. Moreover, the fusion protein approach allows quantitative comparisons of the intrinsic activities of ligands between one single receptor subtype with different Galpha protein subtypes.

Modulation of 5-HT(1A) receptor activation by its interaction with wild-type and mutant g(alphai3) proteins.

Constitutive and agonist-dependent activation of the recombinant human 5-HT(1A) receptor (RC: 2.1.5HT.01A) was investigated by co-expression with a rat G(alphai3) protein in Cos-7 cells. The interaction between the 5-HT(1A) receptor and rat G(alphai3) protein was modulated by substitution of the G(alphai3) protein site for pertussis toxin-catalysed ADP-ribosylation (cysteine(351)) by each of the natural amino acids. Enhanced basal [(35)S]GTPgammaS binding responses (+24 to +189%) were observed with the mutant G(alphai3) proteins containing at position 351 either a histidine, glutamine, serine, tyrosine or a nonpolar amino acid with the exception of a proline. With each of these mutant G(alphai3) proteins, spiperone (10 microM), but not WAY 100635 (10 microM), reduced (-22 to -60%, p<0.05) the enhanced basal [(35)S]GTPgammaS binding response. 5-HT (10 microM)-mediated [(35)S]GTPgammaS binding responses attained for some of the mutant G(alphai3)Cys(351) proteins (Phe, Met, Val and Ala) more than 300% of that obtained with the wt G(alphai3) protein. Similar results were also obtained with the prototypical 5-HT(1A) agonist 8-OH-DPAT and the partial agonist (-)-pindolol. Fusion proteins assembled from the 5-HT(1A) receptor and either the wt G(alphai3)Cys(351), mutant G(alphai3)Cys(351)Gly or G(alphai3)Cys(351)Ile protein displayed similar observations for these ligands as obtained by co-expression of the 5-HT(1A) receptor with each of these G(alphai3) proteins. Both the degree of 5-HT(1A) receptor activation by 8-OH-DPAT and (-)-pindolol, and its inhibition by spiperone, strongly correlate (r(2): 0.78-0.81) with the octanol/water partition coefficients of the mutated amino acid at position 351 of the G(alphai3) protein. The present data also suggest the wt G(alphai3) protein does not result in maximal activation of the 5-HT(1A) receptor by the agonists being investigated.

Real-time analysis of dopamine: antagonist interactions at recombinant human D2long receptor upon modulation of its activation state.

1. Antipsychotic drugs may mediate their therapeutic effects not only by preventing the binding of dopamine but also by decreasing the propensity of the dopamine receptor to assume an active R* state. Ligand-mediated activation and blockade of the recombinant human D(2long) receptor was investigated in CHO-K1 cells upon modulation of its R* state. 2. Both the Ala(371)Lys (A371K) and Thr(372)Arg (T372R) D2long receptor mutants could be activated in a ligand-dependent manner via a chimeric G(alphaq/o) protein, and more efficaciously so than with the promiscuous G(alpha15) protein. 3. Dopamine and partial agonists (E(max): lisuride >> (+)-UH 232 approximately bromerguride) displayed dissimilar Ca(2+) kinetic properties at wild-type and mutant receptors. A371K and T372R D2long receptor mutants demonstrated an attenuated and enhanced maximal response to these partial agonists, respectively. 4. Dopamine antagonists were unable to block the transient high-magnitude Ca(2+) phase at the wild-type D2long receptor upon simultaneous exposure to antagonist and dopamine, while full blockade of the low-magnitude Ca(2+) phase did occur at a later time (onset-time: haloperidol < bromerguride < (+)-butaclamol). A similar, though more efficacious, antagonist profile was also found at the A371K mutant receptor. Conversely, the blockade of the low-magnitude Ca(2+) phase was attenuated (haloperidol) or almost absent [(+)-butaclamol and bromerguride] at the T372R mutant receptor. 5. In conclusion, mutagenesis of the Ala(371) and Thr(372) positions affects in an opposite way the ligand-dependent activation and blockade of the D2long receptor. The observed attenuation of dopamine-mediated Ca(2+) signal generation with different decay-times may underlie distinct properties of the dopaminergic ligands.

Differential signalling of both wild-type and Thr(343)Arg dopamine D(2short) receptor by partial agonists in a G-protein-dependent manner.

G-protein activation and Ca(2+) responses by the wild-type D(2short) receptor and a mutation Thr(343)Arg, in the distal BBXXB motif of its third intracellular loop, were investigated in CHO-K1 cells in terms of ligand:receptor:G-protein interactions. No evidence was obtained for constitutive, agonist-independent receptor activation, but differences in the ligand-mediated activation profiles of both the wild-type and mutant Thr(343)Arg D(2short) receptor were observed. Most of the partial agonists, but not bromocriptine, displayed an enhanced response at the mutant D(2short) receptor, suggesting that the mutation brings the receptor in a more active state. This enhancement was apparent both at the level of G-protein activation ([35S]GTPgammaS binding) and at the effector (Ca(2+) response) and occurred with different G(alpha)-proteins. Partial agonists were also found to act differently via the wild-type D(2short) receptor depending on the involved G(alpha)-protein. Compared with higher efficacy agonists, partial agonists displayed Ca(2+) responses with slower and dissimilar kinetic properties. Lisuride and in particular bromocriptine produced a more potent response in the co-presence of a G(alphao) protein instead of a chimeric G(alphaq/o)- or a promiscuous G(alpha15)-protein. S(+)-propylnorapomorphine showed a similar partial response irrespective of the combined G(alpha)-protein. Bromerguride and (+)-UH 232 induced weak (16 to 21% versus dopamine) intrinsic activity in the co-presence of a G(alphaq/o)-protein in contrast to their silent properties with a G(alpha15)- or a G(alphao)Cys(351)Ile-protein. In conclusion, the present data strongly suggest that multiple activation binding sites are involved with these ligands at the D(2short) receptor, and that their activation may be unravelled by either the mutation or co-expressed G(alpha)-proteins being investigated.

Modulation of ligand responses by coupling of alpha(2A)-adrenoceptors to diverse G(alpha)-proteins.

The hypothesis that different signalling may be mediated via a single alpha(2A)-adrenoceptor (alpha(2A) AR) subtype was investigated by challenging alpha(2) AR ligands in combination with diverse recombinant wt, mutant, and chimeric G(alpha)-proteins. Possible coupling of alpha(2A) AR to endogenous G(alphai/o)-proteins in CHO-K1 cells was excluded by measuring pertussis toxin (PTX)-resistant [(35)S]GTPgammaS-binding responses as a common functional response to alpha(2A) AR activation. (-)-Adrenaline (10 microM) displayed the highest magnitude of [(35)S]GTPgammaS-binding response in the co-presence of a PTX-resistant G(alphao)Cys(351)Ile protein, whereas a decreased response was obtained with the mutant G(alphai1/2)-proteins. Replacement of the last six amino acids at the C-terminal portion of the G(alphao)-protein by the corresponding amino acid region of either the G(alphaz)-, G(alphas)-, G(alphaq)-, or G(alpha15)-protein and co-expression with the alpha(2A) AR resulted in similar maximal (-)-adrenaline-mediated [(35)S]GTPgammaS-binding responses with these chimeric G(alphao)-proteins. The ligands D-medetomidine, BHT 920 (6-allyl-5,6,7,8-tetrahydro-4H-thiazolo[4,5-d]azepin-2-ylamine) and (+)-RX 811059 (2-(2-ethoxy-2,3-dihydro-benzo[1,4]dioxin-2-yl)-4,5-dihydro-1H-imidazole) were weakly active or virtually inactive at the chimeric G(alphao/s)-, G(alphao/q)-, and G(alphao/15)-proteins in contrast to the G(alphao/z)-protein. Furthermore, combining the constitutively active mutant Thr(373)Lys alpha(2A) AR with these chimeric G(alphao)-proteins enhanced the apparent intrinsic activity of d-medetomidine and BHT 920. A similar observation was made using the corresponding fusion proteins, where the stoichiometry of the mutant alpha(2A) AR to the chimeric G(alphao)-protein was fixed at 1.0. These data indicate that a single ligand may display different magnitudes of activation at the alpha(2A) AR subtype coupled to chimeric G(alphao) proteins under controlled conditions of alpha(2A) AR: G(alphao)-protein expression.

Combined scintigraphic and pharmacokinetic investigation of enteric-coated mesalazine micropellets in healthy subjects.

BACKGROUND: There is a growing clinical trend to increase the daily dose of mesalazine, which leads to significant compliance issues associated with multiple dosings of current preparations. AIM: To examine the gastrointestinal performance and systemic exposure of a 1.5 g sachet (micropellets) mesalazine formulation, compared with three enteric-coated tablets (500 mg each, Claversal). METHODS: A randomized, two-way, cross-over pharmacoscintigraphic (scintigraphy plus pharmacokinetics) study and a two-way, cross-over, pharmacokinetic-only study were performed in 24 healthy volunteers (12 subjects per investigation). RESULTS: The relative bioavailability of mesalazine was 92% comparing micropellets with Claversal tablets, and the cumulative urine excretion was c. 26% for both preparations, suggesting comparable systemic exposure for the two types of preparation. In the majority of subjects, drug release from the micropellet formulation occurred predominantly in the terminal ileum and ascending colon. The Claversal tablets disintegrated in comparable intestinal sites, albeit at slightly later time points than the micropellets, principally due to slower gastric emptying for the single-unit formulation. CONCLUSION: The 1.5 g micropellet formulation offers comparable delivery properties to the marketed tablets, but with greater convenience of dosing.

Stimulated [35S]GTP gamma S binding by 5-HT1A receptor agonists in recombinant cell lines. Modulation of apparent efficacy by G-protein activation state.

G-protein activation by different 5-HT receptor ligands was investigated in h5-HT1A receptor-transfected C6-glial and HeLa cells using agonist-stimulated [35S]-GTP gamma S binding to membranes in the presence of excess GDP. 5-HT (10 microM) stimulated [35S]GTP gamma S binding in the C6-glial membrane preparation to a larger extent than in the HeLa preparation; maximal responses with 30 microM GDP were 490 +/- 99 and 68 +/- 12%, respectively. With the 5-HT receptor agonists that were being investigated, the two preparations displayed the same rank order of potency for stimulation of [35S]GTP gamma S binding. In the C6-glial preparation at 0.3 microM GDP, the rank order of maximal effects was: 5-HT (1.00) > 8-OH-DPAT (0.90) = R(+)-8-OH-DPAT (0.87) = 5-CT (0.86) = L694247 (0.84) > S(-)8-OH-DPAT (0.68) = buspirone (0.67) = spiroxatrine (0.67) = flesinoxan (0.64) > ipsapirone (0.53) = (-)-pindolol (0.50) > SDZ216525 (0.25). However, differences in maximal response in the C6-glial preparation were magnified by increasing the GDP concentrations, indicating that the activity state of G-proteins can affect the maximal response. With the exception of 5-CT and L694247, increasing the amount of GDP to 30 microM and higher concentrations resulted in an attenuation of both the ligand's maximal effect (24 to 56%) and apparent potency (6 to 24-fold). Each of the [35S]GTP gamma S binding responses was mediated by a 5-HT1A receptor as indicated by the competitive blockade by WAY100635 and spiperone. Only 5-CT and L694247 in some conditions displayed an efficacy similar to that of 5-HT at the h5-HT1A receptor; the other agents with intrinsic activity are partial agonists at this receptor. The data also suggest that the activity state of the G-proteins is involved in the maximal effects that can be produced by activating the h5-HT1A receptor.

Analysis of ligand activation of alpha 2-adrenoceptor subtypes under conditions of equal G alpha protein stoichiometry.

Variations in the measurement of ligand's intrinsic activity between receptor subtypes is a common consequence of unequal receptor:G protein density ratios. We have investigated ligand activation at the alpha2-adrenoceptor (alpha2-AR) subtypes under defined expression conditions of one receptor molecule for one Galpha protein molecule using fusion proteins. Fusion between either a wt alpha2C AR or a mutant Thr382Lys alpha2C AR and a chimeric Galphaq/il protein displayed robust, transient (-)-adrenaline-mediated Ca2+ responses with similar potencies (pEC50: 7.78 and 7.66) and kinetic properties. A comparison of the intrinsic activities of alpha2 AR agonists found d-medetomidine to be the only compound with an efficacy similar to that of (-)-adrenaline. The Ca2+ responses as mediated by UK 14304, oxymetazoline and clonidine became more potent and efficacious at the Thr381Lys alpha2C AR, whereas the response as mediated by talipexole displayed a higher potency with an unaltered maximal response. Whereas only small differences in ligand's intrinsic activities between the wt alpha2A, alpha2B and alpha2C AR fusion proteins were observed with most ligands, oxymetazoline was virtually silent at the alpha2A AR while active as a partial and apparently full agonist at the alpha2C AR and alpha2B AR, respectively. The mutant alpha2 AR subtypes could be differentiated using the apparent positive efficacy of ligands that used to be defined as antagonists. The following rank order of maximal responses was observed for the Thr381Lys alpha2C AR: idazoxan approximately equals SKF 86466 > atipamezole >> dexefaroxan; Thr373Lys alpha2A AR: SKF 86466 > idazoxan = atipamezole > dexefaroxan; and Thr370Lys alpha2B AR: atipamezole > idazoxan dexefaroxan. RX 811059 (10 microM) was the only compound to be completely silent at both the wt and mutant alpha2 AR subtypes. In conclusion, silent alpha2 AR ligands are probably rare in these specified alpha2 AR systems. Most antagonists may actually possess partial agonist properties at the alpha2 AR subtypes, which are facilitated by the same mutation in the distal portion of their third intracellular loop.

Agonist efficacy at the alpha2A-adrenoceptor:G alpha15 fusion protein: an analysis based on Ca2+ responses.

A fusion protein was constructed between the recombinant human alpha2A-adrenoceptor and a mouse G alpha15 protein to measure the efficacy of agonist-induced Ca2+ responses at a receptor:G alpha15 protein stoichiometry of 1. Activation of this fusion protein in CHO-K1 cells by (-)-adrenaline induced a time- and concentration-dependent (pEC50: 7.28+/-0.04) increase in the intracellular Ca2+ concentration. The magnitude of the Ca2+ response was related to the amount of the fusion protein and the number of surface alpha2A-adrenoceptor binding sites as estimated by [3H]RX 821002 binding. Whereas UK 14304 was as efficacious as (-)-adrenaline, the following ligands displayed partial agonist properties [Emax in percentage vs. (-)-adrenaline: d-medetomidine (76+/-3) > BHT 920 (53+/-3) > clonidine (39+/-4) >> oxymetazoline (10+/-1)]. This ligand activation profile was not affected over a 30-fold range of expression of the fusion protein in contrast to the observed enhancement of the partial agonists' maximal responses by co-expression of the alpha2A-adrenoceptor with increasing amounts of the G alpha15 protein. In conclusion, the fusion protein approach opens perspectives to quantify intrinsic activities of ligands under controlled experimental conditions of a fixed receptor:G alpha15 protein ratio of 1.

Social influences on sexual maturation of female Saguinus oedipus oedipus.

The hypothesis tested was that Saguinus oedipus oedipus females housed with adult males would mature, sexually, at an earlier age than females remaining in their natal family groups. Six females were housed with strange, unrelated males. Five females remained in their natal groups. Blood samples were taken twice weekly, and the plasma was assayed for progesterone. Sexual maturation was operationally defined as that age at which plasma progesterone levels became consistently detectable. Females housing with males did mature at an earlier age than females remaining in their natal groups. In addition, it was noted that the presence or absence of a healthy, reproductive mother in the natal group was not related to the daughter's maturation age. However, whether the natal group, as a whole, inhibited maturation of young females, or an unrelated male accelerated maturation, or both, remains unknown.

Relations among measures of body composition, age, and sex in the common marmoset monkey (Callithrix jacchus).

Few studies of body composition have been done in New World primates. In the study reported here, four methods of assessing body composition (body weight, anthropometry, labeled-water dilution, and total body electroconductivity) were compared in 20 marmosets, aged 0.96 to 7.97 years. Males and females did not differ in any measure (P > 0.05). Body weight ranged from 272 to 466 g, and body fat estimates varied from 1.6 to 19.5%. Strong positive correlations were observed between total body water and total body electroconductivity (R2 = 0.77), body weight and fat-free mass (males R2 = 0.95; females R2 = 0.91), and body weight and fat mass (males R2 = 0.86; females R2 = 0.85; P < 0.01). Male and female slopes were equivalent (P > 0.05) for the regressions of fat and fat-free mass against body weight. Positive correlations also were observed between girth measures and fat-free mass (R2 = 0.48 to 0.78) and fat mass (R2 = 0.60 to 0.74; P < 0.01). A good second- order polynomial relationship was observed between age and fat-free mass for the combined sample (R2 = 0.64). Results indicated that: subjects were lean; there was no sexual dimorphism relative to measures; body weight provided a reliable estimate of fat and fat-free mass; and within-subject body weight changes reflected a similar relationship between body weight and fat-free mass as did that across subjects.

The importance of porcine sperm parameters on fertility in vivo.

It would be desirable to use semen parameters to predict the in vivo fertilizing capacity of a particular ejaculate. In animal production, an ejaculate is divided into multiple doses for artificial insemination (AI); therefore, it would be economically beneficial to know the functional quality (i.e., fertility) of the semen before it is inseminated. To identify a predictive assay of the fertilizing capacity of a porcine ejaculate, we performed 4 rapid assays of sperm quality (motility, viability, physiological status as assessed by chlortetracycline fluorescence, and ATP content) on samples from 9 ejaculates, before and after a thermal stress test (42.5 degrees C, 45 min). These parameters were subsequently correlated with in vivo fertility resulting from AI with 2 sperm doses, 3 x 10(9) or 0.3 x 10(9) motile cells in 70 mL (optimal or suboptimal sperm number per insemination, respectively) from these same ejaculates. No parameter was correlated to the fertility rates obtained after inseminating with the optimal semen doses, either before or after the thermal stress test (P > 0.05). However, with respect to the animals inseminated with the suboptimal semen dose, sperm motility (the percentage of motile spermatozoa as assessed visually by microscopy) prior to thermal stress was well-correlated to fertility rates (r = 0.783, P = 0.01). The percentage of spermatozoa displaying the chlortetracycline Pattern AR (acrosome reaction) was also statistically related to fertility (r = 0.05, P = 0.04), but the biological importance of this relationship is questionable given the small variation among ejaculates (range: 0 to 2%). No other sperm parameter was significantly related to fertility rates in this group (P > 0.05). These data, therefore, indicate that sperm motility is a useful indicator of sperm fertilizing capacity in vivo. Moreover, to identify a predictor of semen fertility it is critical that the number of spermatozoa used during insemination is sufficiently low to detect differences in sperm fertilizing efficiency.

The effect of heparin on motility parameters and protein phosphorylation during bovine sperm capacitation.

It is generally accepted that incubation with heparin is required to induce capacitation of ejaculated bovine spermatozoa in vitro. The capacitation process implicates many biochemical events, and is correlated with modified sperm motility and the phosphorylation of specific proteins on tyrosine residues. To better understand the molecular basis of heparin-induced capacitation, bovine spermatozoa were incorporated with a radioactive substrate of protein kinases [gamma32P]-ATP, to observe protein phosphorylation dynamics over time. Sperm motion parameters including the percentage of motile spermatozoa, amplitude of lateral head displacement (ALH) and flagellar beat cross frequency (BCF) were assessed to determine whether the protein phosphorylation patterns induced by heparin also promote changes in motility. Capacitation was confirmed using the chlortetracycline fluorescence assay and the appearance of 'pattern B' stained spermatozoa. Evaluation of the different motility parameters during capacitation reveal that heparin has a marked negative effect, over time, on the percentage of motile spermatozoa, consistent with hyperactivation. Indeed, the presence of heparin greatly increases the BCF of bull spermatozoa and induces a significant increase in the ALH compared to spermatozoa incubated without heparin. We detected several sperm proteins that are phosphorylated over time. A 45 kDa protein is the most intensely phosphorylated of the sperm proteins. However, it is visible regardless of the presence of heparin. Interestingly, a second phosphorylated protein of approximately 50 kDa undergoes more intense phosphorylation with heparin than without. In summary, the present study demonstrated that heparin induces physiological changes in several sperm motility parameters including ALH, BCF and the percentage of motile spermatozoa. Heparin also increases the intensity of phosphorylation of a 50 kDa sperm protein. These results suggest that capacitation of bovine spermatozoa and capacitation-associated motility changes may be regulated by a mechanism that includes protein phosphorylation, and that a presently unknown protein kinase is involved.