Total Binding Capacity Research Articles

Immobilised metal ion affinity chromatography purification of alcohol dehydrogenase from baker’s yeast using an expanded bed adsorption system

Alcohol dehydrogenase (ADH) from solutions of homogenised packed bakers’ yeast has been successfully purified using immobilised metal-ion affinity chromatography in an expanded bed. Method scouting carried out using pure ADH solutions loaded onto 5-ml HiTrap columns charged with Zn 2+, Ni 2+ and Cu 2+ and eluted using 0–50 m M EDTA gradient found that charging with Zn 2+ gave the highest recovery and the lowest EDTA concentration required for elution. These results were used to develop a protocol for the expanded bed system and further tested using clarified yeast homogenate loaded onto XK16/20 packed beds (approximately 30 ml) packed with Chelating Sepharose FastFlow matrix in order to determine the optimum elution conditions using EDTA. The ADH was found to elute at 5 m M EDTA and the dynamic and total binding capacities of Streamline chelating for ADH were found to be 235 U/ml and 1075 U/ml matrix, respectively. Expanded bed work based on a step EDTA elution protocol demonstrated that ADH could be successfully eluted from unclarified homogenised bakers’ yeast diluted to 10 mg/ml total protein content with a recovery of 80–100% that was maintained over five consecutive runs with a vigorous clean-in-place procedure between each run.

Interaction between leukocyte elastase and elastin: quantitative and catalytic analyses

Solubilization of elastin by human leukocyte elastase (HLE) cannot be analyzed by conventional kinetic methods because the biologically relevant substrate is insoluble and the concentration of enzyme–substrate complex has no physical meaning. We now report quantitative measurements of the binding and catalytic interaction between HLE and elastin permitted by analogy to receptor–ligand systems. Our results indicated that a limited and relatively constant number of enzyme binding sites were available on elastin, and that new sites became accessible as catalysis proceeded. The activation energies and solvent deuterium isotope effects were similar for catalysis of elastin and a soluble peptide substrate by HLE, yet the turnover number for HLE digestion of elastin was 200–2000-fold lower than that of HLE acting on soluble peptide substrates. Analysis of the binding of HLE to elastin at 0°C, in the absence of significant catalytic activity, demonstrated two classes of binding sites ( K d=9.3×10 −9 M and 2.5×10 −7 M). The higher affinity sites accounted for only 6% of the total HLE binding capacity, but essentially all of the catalytic activity, and dissociation of HLE from these sites was minimal. Our studies suggest that interaction of HLE with elastin in vivo may be very persistent and permit progressive solubilization of this structurally important extracellular matrix component.

Development of a Homologous Radioimmunoassay for Mouse Growth Hormone Receptor*

A RIA for mouse GH receptor (mGHR) was developed. A synthetic peptide corresponding to the carboxyl-terminal 14 amino acids of the mGHR (GHR-2 peptide) was used as the antigen for antiserum production. The synthetic peptide was also used as the standard and radioligand in the RIA. The ability of the antiserum to recognize the mGHR was demonstrated by quantitating receptor concentrations in liver and mammary gland from virgin and 15-day-pregnant mice. Serial dilutions of these samples yielded displacement curves parallel to the synthetic peptide. No significant cross-reactivity was seen with serum from virgin or 15-day-pregnant mice, mGH, recombinant mGH-binding protein (mGHBP), a synthetic peptide identical to the hydrophilic tail of mGHBP, or a 14-amino acid synthetic peptide corresponding to amino acids 338-351 of mGHR (GHR-1 peptide). The concentration range of the mGHR RIA was 0.5-200 nM, and the intra- and interassay coefficients of variation were 6.5% and 6.1%, respectively. The concentration of liver GHR increased significantly during pregnancy compared with that in virgin mice, from 0.246 +/- 0.045 pmol/mg protein (mean +/- SEM; n = 5) in the virgin animals to 1.015 +/- 0.159 pmol/mg protein (n = 5) in pregnant mice. In contrast, the mGHR concentration in the mammary gland decreased significantly during pregnancy from 0.606 +/- 0.201 pmol/mg protein (mean +/- SEM; n = 5) to 0.299 +/- 0.027 pmol/mg protein (n = 5). Comparison of the total number of binding sites in livers from virgin and pregnant mice using the GH RRA and the combined results of the mGHR and mGHBP RIAs showed that the two methods gave almost identical results for livers from virgin animals, or 0.363 +/- 0.063 pmol/mg protein (mean +/- SEM; n = 3) and 0.371 +/- 0.008 pmol/mg protein (n = 3) for the GH RRA and the mGHR plus mGHBP RIAs, respectively. However, in livers from pregnant animals, the combined results from the mGHR and mGHBP RIAs were approximately 1.8 times higher than those obtained by the GH RRA, or 6.732 +/- 0.612 pmol/mg protein (mean +/- SEM; n = 3) and 3.693 +/- 0.67 pmol/mg protein (n = 3) for the mGHR plus the mGHBP RIAs and the GH RRA, respectively. The increase in the total GH binding capacity in livers from pregnant mice compared with those from virgin animals was largely due to an increase in the GHBP content. The increase in GHR was only 2.4-fold, or from 0.153 +/- 0.01 pmol/mg protein (mean +/- SEM; n = 3) in virgin mice to 0.364 +/- 0.03 pmol/mg protein (n = 3) in the 15-day-pregnant mice, whereas GHBP increased almost 30-fold during pregnancy, or from 0.218 +/- 0.003 pmol/mg protein (mean +/- SEM; n = 3) in virgin animals to 6.369 +/- 0.607 pmol/mg protein (n = 3) in pregnant mice.

Effect of oleoyl-estrone administration on corticosterone binding to tissues of lean and obese Zucker rats

A group of female Zucker lean and obese rats was treated with 3.5 μmol/day kg of oleoyl-estrone in liposomes (OE) injected i.v. continuously for 14 days with inserted osmotic minipumps. Samples of liver were extracted on days 0, 3, 6, 10 and 14 and the expression of corticosterone-binding globulin (CBG) was determined by Northern blot. On the same dates, the total binding capacity of plasma, liver, periovaric white adipose tissue (WAT) and subcutaneous WAT was also determined using tritium-labelled corticosterone. Treatment with OE resulted in diminished CBG gene expression in the liver, this being more marked in the obese rats. Basal (time 0) corticosterone binding was higher in the plasma, liver and WAT of lean rats. Treatment with OE resulted in a gradual and general loss of binding capacity in the plasma and all tissues studied, for lean and obese rats alike. Since CBG decreases may result in enhanced glucocorticoid availability (and effects), the global decrease in corticosterone binding observed can be interpreted as a counteractive response to the energy imbalance elicited by OE.

Bovine adrenal glomerulosa cells express such a low level of functional B2 receptors that bradykinin does not significantly increase their aldosterone production.

It was recently demonstrated that bradykinin (BK) stimulates aldosterone secretion in bovine adrenal glomerulosa (BAG) cells. The aim of the present study was to characterize the mechanism of action of BK on these cells. Binding experiments with the radioligand 125I-[Tyr8]BK revealed the presence of a relatively small amount (Bmax = 180 +/- 55 fmol/mg of protein) of high affinity (Kd = 0.65 +/- 0.17 nM) binding sites. BK induced a time- and concentration-dependent increase of [3H]inositol trisphosphate ([3H]IP3) in myo-[3H]inositol-labeled BAG cells. A maximal response was obtained with 10 nM BK and the EC50 value was 1.0 +/- 0.5 nM. 125I-[Tyr8]BK binding and BK-induced IP3 production were inhibited by the selective B2 receptor antagonist Icatibant (1 microM) and unaffected by the selective B1 receptor antagonist [DesArg9, Leu8]BK (1 microM). In fura-2 loaded BAG cells, BK (100 nM) induced a typical biphasic Ca2+ response composed of a rapid and transient increase of intracellular Ca2+ concentration [Ca2+]i which slowly declined to a level that remained above basal level for about 5 min. In the presence of EGTA (2 mM), the rapid and transient calcium increase was unaffected whereas the plateau phase was abolished. Angiotensin II (Ang II, 100 nM) also elicited a typical biphasic response in BAG cells. However the rapid and transient elevation of [Ca2+]i was followed by a sustained plateau phase which remained above the basal level for more than 10 min. Although BAG cells express functional B2 receptors, no secretion of aldosterone was observed after stimulation with 100 nM BK for 120 min. Under the same conditions Ang II increased by about 10-fold the basal level of aldosterone. The lack of effect of BK is probably attributable to its very transient effect on IP3 production. Pretreatment of BAG cells with 100 nM BK for 20 min reduced by 70 +/- 10% their total binding capacity. These results suggest a rapid and very efficient desensitization process. We conclude that BAG cells express functional B2 receptors. The weak production of second messengers and the rapid desensitization process could explain why BK fails to increase aldosterone production in these cells. Since functional B2 receptors are expressed in BAG cells it is likely that under some specific physiological or pathological conditions these receptors may play a significant role in aldosterone secretion. However these conditions remain to be determined.

Beta receptor isoforms are not essential for thyroid hormone-dependent acceleration of PCP-2 and myelin basic protein gene expression in the developing brains of neonatal mice

In rat pups, thyroid hormone dependent brain development coincides with the appearance of the thyroid hormone receptor (TR) β1 isoform. This finding led to the suggestion that TR β1 plays an essential role in brain development. The recent availability of a mouse TR β knockout strain allowed us to test this possibility by determining whether TR β is essential for the normal developmental pattern of expression of two thyroid hormone regulated brain genes, myelin basic protein (MBP), and Purkinje cell protein 2 (Pcp-2). Northern analysis of total mRNA from the brains of wild-type mice established that, as in the rat pup, the initial rate of rise of the MBP and Pcp-2 mRNA is slowed in the hypothyroid state. Supporting the effectiveness of TR β gene deletion was the finding that the thiiodothyronine (T3) nuclear binding capacity in the livers and brains of knockout animals was consistent with the fractional contribution of TR β1 to total binding capacity in the wild-type tissues. Further, no TR β1 could be detected by isoform-specific immunoprecipitation of nuclear receptor extracts. However, deletion of the functional TR β in the TR β knockout mice did not affect the normal ontogeny of expression of the Pcp-2 and MBP genes in the postnatal pup. We conclude that TR β is not essential for the normal developmental expression of these T3 dependent brain genes.

Is Cortisol the Aging Hormone

The “Dorian Gray” senescence of several fish species and a small Australian marsupial mouse (Antechinus), is well documented to be caused by a large increase of cortisol triggered by stress. While cortisol is important in response to trauma, infection, exercise, anxiety, depression and up to 10-fold during surgery, it may also cause involution of the thymus, depression of immune response, tissue damage, fat deposition, confusion and dementia. Excess cortisol is transported by the cortisol binding globulin (CBG) and by albumin. The specific affinity for albumin is lower, but the total binding capacity over 1000-fold higher (42g/L = >200,000ug/L), than CBG (0.037g/L = 250ug/L). With increasing cortisol levels, CBG becomes saturated and a larger proportion of cortisol is carried by albumin. Synthetic glucocorticoids and other hormones are also albumin-bound. In fish and Antechinus the level of albumin is low, perhaps explaining why excess cortisol can cause rapid aging, associated with immune depression. In premature infants, hypoal-buminemia and in the elderly, albumin is decreased and the proportion of free steroid increased during stress. This factor may help explain why the level of serum albumin has emerged in major studies as an important biochemical factor in determining all-cause mortality and morbidity. One area that has shown the possibility of achieving high albumin profiles (55g/L, A/G ratio 2.75), is a hygiene regimen. Hygiene reduces globulin levels, allowing higher albumin levels, while maintaining total protein. We have observed very high albumin profiles (59g/L and an A/G ratio 4.13), associated with the use of a hygiene regime in a few cases.

Effects of propofol and pentobarbital on ligand binding to GABAA receptors suggest a similar mechanism of action

The GABAA receptor is allosterically modulated by a number of anesthetics and barbiturates. We have examined the effects of propofol and pentobarbital on the binding of the receptor agonist [3H]muscimol and the benzodiazepine modulators [3H]flunitrazepam and [3H]Ro15-4513 to bovine brain membranes. Both agents potentiated the binding of [3H]muscimol (5 nM), with EC50 values of 18.7 and 276 microM, respectively. The binding of [3H]muscimol is heterogeneous, suggesting the presence of both high (Kd approximately 10 nM) and low (Kd approximately 0.1-1.0 microM) affinity sites. The major effect of both propofol and pentobarbital was to increase the affinity of the lower affinity sites without changing the total binding capacity. In contrast, the steroid anesthetic alphaxalone did not affect the affinity of these sites, suggesting that this drug has distinct effects on the GABAA receptor. Propofol and pentobarbital also increased the binding of the benzodiazepine agonist [3H]flunitrazepam and decreased the binding of the inverse agonist [3H]Ro15-4513. The results of these studies demonstrate that propofol and pentobarbital modulate the binding of ligands to the GABAA receptor in a similar manner, suggesting that these drugs may have a common mechanism of action.

Characterization, Guanosine 5′-O-(3-Thiotriphosphate) Modulation, Daily Variation, and Localization of Melatonin-Binding Sites in the Catfish (Silurus asotus) Brain

Characteristics, guanosine 5′-O-(3-thiotriphosphate) (GTPγS) modulation, daily variation, and localization of melatonin-binding sites in the brain of a nocturnal teleost, the catfishSilurus asotus,were studied by radioreceptor assay using 2-[125I]iodomelatonin as the radioligand. The specific binding was rapid, stable, saturable, and reversible. The radioligand binds to a single class of receptor site with an affinity (Kd) of 30.7 ± 7.3 pMand total binding capacity (Bmax) of 9.76 ± 0.79 fmol/mg protein (mean±SE,n=5). The binding sites were highly specific for 2-iodomelatonin and melatonin. The specificity was almost identical to that of functional melatonin receptors in the dermal and epidermal melanophores in this species and that of ML-1 subtype melatonin receptors in vertebrates, including melatonin-binding sites in the goldfish brain. GTPγS treatment altered both theKdandBmaxvalues, indicating that melatonin-binding sites in the catfish brain are coupled to G protein. TheBmaxvalues exhibited no daily variation under light–dark cycles of 12 hr light:12 hr dark whereas plasma melatonin levels andKdfluctuated in a rhythmic fashion. The density of melatonin-binding sites in discrete brain areas was determined to be highest in optic tectum–thalamus and hypothalamus, intermediate in telencephalon, cerebellum, and medulla oblongata, and lowest in olfactory bulbs. These results suggest that melatonin secreted from the pineal organ and/or retina plays neuromodulatory roles in the catfish brain via G protein-coupled melatonin receptors. Characteristics of melatonin receptors seem to be highly conserved during evolution, although the density of melatonin receptors is not regulated by melatonin itself in this species.

Serum trypsin-inhibitory activity in five species of farmed fish

The trypsin-inhibitory activity of the serum of five cultured fish species, halibut (Hippoglossus hippoglossusL.), turbot (Scophthalmus maximusL.), arctic char (Salvelinus alpinusL.), rainbow trout (Oncorhynchus mykiss, Walbaum) and brown trout (Salmo truttaL.) was investigated. Individual fish were bled and the serum trypsin-inhibitory activity analysed using an arginine-aniline dye ester as colorimetric substrate. Results for the five species indicate that levels of α2-macroglobulin-like activity decrease significantly (P<0·05) from halibut through turbot, brown trout, char and finally rainbow trout with the lowest levels. Analysis of the total trypsin binding capacity indicates a ranking with decreasing concentration of char, turbot, halibut, brown trout and rainbow trout. Significant differences occurred only in certain cases, brown and rainbow trout were significantly (P<0·05) lower than char, and rainbow trout was significantly (P<0·05) lower than turbot. The results indicate that halibut and turbot possess a higher level of α2-macroglobulin-like activity than the other species in the study, whereas char had the highest total trypsin-inhibitory level.

Lipophorin: A hemolymph juvenile hormone binding protein in the german cockroach, Blattella germanica

We examined the binding of [ 3H](10 R) juvenile hormone (JH) III to lipophorin that was purified from the hemolymph of Blattella germanica. Binding was found to be specific, saturable and with high affinity to JH III. Using Scatchard analysis, the equilibrium dissociation constant (K d) and total binding capacity (B max) were estimated to be 9.75±0.64 nM and 0.241±0.02 nmol/mg protein, respectively. Competitive displacement studies with racemic JH III, JH I, cuticular hydrocarbon, contact sex pheromone, and the JH analogs pyriproxyfen, fenoxycarb, and hydroprene showed that only JH III readily displaced [ 3H](10 R)JH III from the binding site. However, hydroprene competed for the JH III binding site more effectively than the other two JH analogs. Photoaffinity labelling using the JH III analog [ 3H]epoxyfarnesyl diazoacetate demonstrated that the JH binding site was on apolipophorin-I, the large subunit of the lipophorin complex.

Interaction of [3H]orphanin FQ and 125I-Tyr14-orphanin FQ with the orphanin FQ receptor: kinetics and modulation by cations and guanine nucleotides.

The heptadecapeptide orphanin FQ (OFQ) has been identified as the endogenous ligand for a G protein-coupled receptor (OFQ-R), which, despite its high degree of sequence similarity to opioid receptors, fails to bind opioid ligands. We developed two radioligands for the OFQ-R: a tritiated native OFQ peptide ([3H]OFQ) and a radioiodinated form in which Leu14 was substituted by tyrosine (125I-Tyr14-OFQ). Their binding properties were examined in human embryonic kidney (HEK) 293 and Chinese hamster ovary (CHO) cells heterologously expressing the OFQ-R at different levels (HEK 293 expressed 40-fold more OFQ-R than did CHO). Both ligands exhibited rapid, monophasic association kinetics in each cell line. Dissociation of both ligands from OFQ-R expressed in HEK 293 cells was biphasic, whereas dissociation of 125I-Tyr14-OFQ from OFQ-R expressed in CHO cells was monophasic and slow. Saturation binding analysis revealed two affinity states in HEK 293 cells with binding parameters in accord with those determined kinetically. In CHO cells, 125I-Tyr14-OFQ detected a single affinity state with an intermediate Kd value of 54 pM. Optimal binding of the radioligands required 1-5 mM MgCl2, whereas millimolar concentrations of ZnCl2, CaCl2, MnCl2, and NaCl reduced specific binding of both ligands. A nonhydrolyzable GTP analog [guanosine-5'-(beta,gamma-imido)triphosphate] reduced the affinity of both OFQ ligands to their receptor without significant changes in the total binding capacity, indicating functional interactions between the OFQ-R and G proteins. In rat brain membranes, specific, saturable binding of 125I-Tyr14-OFQ was demonstrated to be pharmacologically identical to the heterologously expressed OFQ-R. Taken together, these results indicate that 125I-Tyr14-OFQ and [3H]OFQ exhibit virtually identical characteristics and are suitable for the pharmacological analysis of the OFQ-R.

Melatonin Binding Sites in the Goldfish Retina

Melatonin binding sites in the goldfish retina were characterized by radioreceptor assay using 2-[125|]iodomelatonin as the radioligand. The specific binding to goldfish retinal membranes is rapid, stable, saturable and reversible. Saturation studies demonstrated that 2-[125|]iodomelatonin binds to a single class of sites with an affinity constant (Kd) of 61.9 ± 5.7 pM, a total binding capacity (Bmax) of 6.52 ± 0.79 fmol/mg protein and Hill coefficients (nH) of 1.07 ± 0.03 (mean ± SEM, n = 6). Competition experiments with various indoles and neurotransmitters revealed the following order of affinities: 2-iodomelatonin > melatonin > 6-hydroxymelatonin > 5-methoxytryptamine = N-acetylserotonin > 5-methoxytryptophol. The other indoles and neurotransmitters tested were much less effective. The order resembles with those reported for the goldfish brain and the ML-1 subtype melatonin receptors in vertebrates. Co-incubation of retinal membranes with a non-hydrolyzable GTP analog, guanosine 5′-O-(3-thiotriphosphate), significantly reduced the specific binding. These results suggest that in the goldfish, ocular melatonin plays neuromodulatory roles in the retina via G protein-coupled melatonin receptors with picomolar affinity.

The influence of clinical status on total bilirubin binding capacity in newborn infants

The influence of clinical status on total bilirubin binding capacity in newborn infants

3H]Nemonapride Binding in Human Caudate and Putamen

The binding of [3H]nemonapride to human postmortem caudate and putamen tissue was autoradiographically investigated using several antipsychotic drugs. Saturation experiments revealed a single population of binding sites (dissociation constant (KD) 0.38 ± 0.01 nM, and total binding capacity (BMAX) 55 fmol/TE). Prototypic dopamine (DA) receptors antagonists displaced [3H]nemonapride in a monophasic manner. The order of displacement potency was expected for DA D2-like receptors: spiperone > (+)butaclamol ≥ chlorpromazine > (−)sulpiride > ketanserin. Displacement with serotonergic antagonists suggests that in human caudate and putamen tissue [3H]nemonapride may have a very low affinity serotonergic component. However, [3H]nemonapride displays a high affinity and selectivity for DA D2-like receptors and should make it a preferred compound for tritium-based autoradiography.

Binding of phosphate and sulfate anions by purine nucleoside phosphorylase from E. coli: ligand-dependent quenching of enzyme intrinsic fluorescence

Steady-state and time-resolved emission spectroscopy was applied to a study of the binary and ternary complexes of pure E. coli purine nucleoside phosphorylase (PNP) with phosphate (P i; a substrate) and a close non-substrate analogue (sulfate; SA). The quenching of enzyme fluorescence by P i was bimodal, best described by two modified Stern-Volmer equations fitted independently for “low” (below 0.5 mM P i) and “high” (above 0.5 mM P i) ligand concentrations. At P i > 0.5 mM, binding is characterized by a fortyfold higher dissociation constant ( K d2 = 1.12 ± 0.10 mM), i.e. by a lower affinity for phosphate, with a sevenfold lower quenching constant and 1.6-fold higher accessibility. By contrast, the binding of SA, and the resultant fluorescence quenching, was unimodal, with K d = 1.36 ± 0.07 mM, comparable to the K d for P i at “high” P i, with a total binding capacity of one sulfate or phosphate group per enzyme subunit. SA proved to be a competitive inhibitor of phosphorolysis with K i = 1.2 ± 0.2 mM vs. P i, hence similar to its K d. SA at a concentration of 5 mM did not affect the P i affinity at P i < 0.5 mM, but led to a reduced affinity and twofold higher P i binding capacities at P i > 0.5 mM. The resultant fluorescence quenching by P i decreased at 5 mM SA, with lower Stern-Volmer constant ( K SV) and fractional accessibility ( f a) values. Increasing concentrations of P i reduced the enzyme affinity for SA, characterized by a higher K d. The Hill model showed negative cooperative binding of P i in the absence and presence of 5 mM SA with Hill coefficients h = 0.60 ± 0.01 and h = 0.83 ± 0.07, respectively. SA exhibited non-cooperative binding in the absence of P i ( h = 1.08 ± 0.01) and negative cooperative binding in the presence of P i ( h < 1). PNP fluorescence decays were best fitted to a sum of two exponentials, with an average lifetime of 2.40 ± 0.14 ns, unchanged on interaction with quenching ligands, and pointing to static quenching. The overall results are relevant to the properties of PNP from various sources, in particular to the design of potent bisubstrate analogue inhibitors.

Dietary folic acid, uterine function and early embryonic development in sows

The present study was designed to determine the role of folic acid on uterine environment and embryonic development during early gestation in the pig. Thirty-two, third parity, crossbred sows received a diet supplemented with 0 or 15 mg kg−1 of folic acid. The treatments started 2 wk before expected estrus and lasted until slaughter on either day 12 or 15 after mating. One uterine horn was used to collect conceptuses and uterine "flushings" for hormonal and metabolite determinations; conceptuses from the other horn were enzymatically dispersed and placed in cell culture with and without dehydroepiandrosterone (DHEA). The decrease in serum folates was attenuated (P ≤ 0.06) and the total and saturated folate binding capacities in early gestation were increased (P &lt; 0.01) in sows receiving additional dietary folic acid. The volume of uterine flushings recovered was greater (P ≤ 0.02) on day 15 than on day 12, as was its content of protein (P ≤ 0.06). In sows receiving the dietary supplement of folic acid, total uterine prostaglandin (PG)E2 was three times higher on day 12 and two times higher on day 15 (P &lt; 0.04) than for sows fed the experimental diet without supplement; although numerically substantial (60% higher), the effect was not significant for PGF2α (P ≥ 0.16). Conceptus homogenates contained more folic acid (P ≤ 0.02) and DNA (P ≤ 0.0001) on day 15 than on day 12. Their total protein content, in sows slaughtered on day 12 of gestation, tended (P ≤ 0.07) to be higher in supplemented than in unsupplemented animals. The synthesis of estradiol-17β by the conceptus cells, used as an index of embryonic maturity, tended (P ≤ 0.07) to be lower for treated than untreated sows, especially in conceptus cell culture without DHEA. Therefore, the improvement in embryonic survival attributed to dietary supplements of folic acid might be linked to changes on the secretion of uterine prostaglandins and possibly on embryonic development. Key words: Folic acid, uterus, embryo, sow

Non-competitive anti-oestrogenic actions of progesterone antagonists in primate endometrium: enhancement of oestrogen and progesterone receptors with blockade of post-receptor proliferative mechanisms.

Previous studies have shown that the progesterone antagonists (antiprogestins) inhibit oestrogen-dependent endometrial proliferation in ovariectomized monkeys, without having affinity to the oestrogen receptor (ER). This study was designed to investigate the effect of the antiprogestins mifepristone (RU 486) and onapristone (ZK 98,299), on the concentration of ER and progesterone receptor (PR) in the endometrium of long-term ovariectomized cynomolgus monkeys (Macaca fascicularis). In untreated monkeys, tissue preparations bound in total 228 +/- 68 pmol [3H]-oestradiol/g protein (ER), and 119 +/- 42 pmol [3H]-R5020/g protein (PR). These values were not significantly different from the total binding capacities of tissues from monkeys treated with RU 486 alone or primates treated with oestradiol plus progesterone. Treatment with oestradiol alone almost doubled the ER and PR concentrations. Combined treatment with oestradiol and RU 486 enhanced the ER and PR concentrations in a dose-dependent manner: 1 mg/kg body weight (bw) RU 486/kg increased both ER and PR contents about 3-fold. The dose of 5mg/kg bw RU 486 or onapristone increased the ER and PR concentrations almost 6- and 5-fold respectively, compared with the oestradiol-treated controls. Our results demonstrate that RU 486 and onapristone increased the endometrial ER and PR concentrations far beyond the physiological level in ovariectomized, oestradiol-treated monkeys. Whether the over-expression of ER and PR in the presence of antiprogestins is causally related to the antiproliferative impact of antiprogestins in the endometrium (non-competitive anti-oestrogenic effects) or is an independent action in unknown.

Copper(II) binding abilities of molecular weight fractionated humic acids and their mixtures

It was found that the intermolecular interaction between molecular weight fractionated humic acids (HAs) affected the copper(II) binding abilities. The mean values of copper(II) binding constants ( μ av ) and the total binding capacities ( N t ) of the molecular weight fractionated HAs were in the range of 6.0–6.6 and 4.7–5.4 (meq g −1 of C), respectively. The μ av and N t values of the unfractionated HA, which could be regarded as a mixture of the fractions, were smaller than those of the fractionated ones (5.6 and 3.8). Moreover, when the fractionated HAs were mixed in the various mixing ratio, the μ av and N t values of all mixtures were not identical to arithmetic mean values of the fractions, but were smaller than those values of the fractions (5.5–5.7 and 2.2–3.6). These results showed the decrease of the copper(II) binding abilities by mixing the fractions. In order to explain these results, the UV-visible absorption, fluorescence, Fourier transform infrared spectra and gel permeation chromatogram of the fractionated HAs and their mixtures were investigated. From the obtained results, we concluded that the decrease of binding abilities in the mixtures was due to the shielding effect of the copper(II)-binding sites by intermolecular interaction between the molecular weight fractions.

Thyroid hormone receptor isoform content in cultured type 1 and type 2 astrocytes.

Immunohistochemical studies previously reported from this laboratory showed that astrocytes in adult rat brain appear devoid of all thyroid hormone receptor (TR) isoforms. These findings, however, contrast with reports of measurable nuclear T3 binding in astrocytes in cell culture. To address this discrepancy, TR protein and messenger RNA (mRNA) content of type 1 and type 2 astrocytes in culture were assayed. Type 1 cells represent astrocytes present in brain in vivo. Type 2 astrocytes differentiate in culture from bipotential progenitor O-2A cells in the presence of serum. Under serum-free conditions, these progenitor cells differentiate into oligodendroglia. Total nuclear T3 binding capacity in both type 1 and type 2 astrocytes was approximately 3000 sites/cell. Northern blots showed the presence of mRNA for TRbeta1, TRalpha1, and TRalpha2 in type 2 cells but failed to reveal the presence of these mRNAs in type 1 astrocytes. Moreover, Northern blots also failed to reveal TRbeta2 mRNA in both type 1 and type 2 astrocytes. These findings, therefore, raised a question as to which receptor isoform was responsible for the nuclear binding capacity observed in type 1 astrocytes. As anticipated, immunocytochemical analysis demonstrated prominent nuclear signals for TRbeta1, TRalpha1, and TRalpha2 mRNA in type 2 astrocytes but failed to demonstrate TRbeta1, TRalpha1, or TRalpha2 in type 2 astrocytes. Application of RT-PCR, however, revealed the presence of low levels of TRbeta2 mRNA in type 1 astrocytes. When stained with a specific anti-TRbeta2 antiserum, both type 1 and type 2 astrocytes showed a strong fluorescent signal concentrated in the nucleus. These data indicate that under the special conditions of cell culture, expression of the TRbeta2 isoform in type 1 accounts for the measured nuclear T3 binding capacity.