Immunological identification of two female-specific proteins from the plasma of Indian freshwater murrel,Channa punctatus (Bloch)

Existence of a non-phosphorylated female-specific protein (FS II), in addition to phosphorylated vitellogenin (FS I), in the plasma of murrel by exogenous administration of estradiol-17beta is reported. Polyspecific rabbit antibodies were raised against estrogen-inducible murrel plasma proteins. This antiserum was absorbed with normal male serum in order to obtain female-specific antiserum (FSAS). Radial immunodiffusion studies suggested that both the proteins (FS I and FS II) were present in the plasma of E2-treated and normal vitellogenic females and in the ovarian homogenate from gravid females, but absent in normal male plasma. Autoradiographic experiments demonstrated that phosphorus moiety was attached with FS I only. Further, immunoelectrophoretic analysis and peptide maps supported the observation that FS I and FS II were discrete, unrelated female-specific proteins.     

A rapid specific radioimmunoassay for unconjugated estriol in plasma.

A rapid, non-chromatographic radioimmunossaay for unconjugated estriol in pregnancy plasma has been developed which utilizes a commonly available antiestrogen antisera. Estradiol-17beta and estrone demonstrate 135% relative cross-reactivity with our antiserum, as compared with 100% for estriol. Specificity is achieved by purification of estriol with solvent partitioning using benzene: petroleum ether (1:1). The results obtained using this method are similar to a radioimmunoassay utilizing a highly specific, but commercially unavailable, antiestriol antiserum. The method is precise, with coefficients of variation ranging from 3.0 to 8.2%.     

In vivo evidence that the rise in plasma IL 6 following injection of a fever-inducing dose of LPS is mediated by IL 1 beta

Although it has often been speculated that Interleukin (IL) 1 alpha and IL 1 beta are circulating endogenous pyrogens (EP), there are few data demonstrating an elevation of these cytokines in the plasma of febrile animals. We hypothesized that IL 1 is released locally and may act to stimulate the release of another pyrogen, IL 6, which circulates to the brain to cause fever. The major purpose of the present study was to determine whether pretreatment of rats with antiserum to IL 1 beta, which attenuates lipopolysaccharide (LPS) induced fever, also results in an attenuation of the rise in plasma and cerebrospinal fluid (CSF) concentrations of IL 6. Our results show that injection of IL 1 beta produced dose-dependent rises in temperature and increases in plasma and CSF IL 6 activity, and that pretreatment of rats i.v. with antiserum to IL 1 beta produced a 55% decrease in the fever caused by LPS injection, a 68% decrease in plasma IL 6, and a 67% decrease in CSF IL 6. These data confirm the findings of previous studies that IL 1 beta is required for a portion of LPS-induced fever and also provide the first in vivo demonstration that the rise of IL 6 in rats injected with a fever-inducing dose of LPS can be significantly blocked by antiserum to IL 1 beta. Overall, the data in our study can be interpreted as being consistent with the hypothesis that the pyrogenic effect of IL 1 beta is mediated mainly through the release of IL 6, but conclusive confirmation of this hypothesis must await studies with antibodies to IL 6.     

Engineered recombinant factor VII Q217 variants with altered inhibitor specificities.

Recombinant factor VII with residue 217 (chymotrypsinogen numbering system) converted to alanine (VIIQ217A), glutamic acid (VIIQ217E), or glycine (VIIQ217G) was characterized. In a prothrombin time assay, VIIQ217E demonstrated 100%, VIIQ217A 15%, and VIIQ217G <1% clotting activities relative to wild-type VII. Binding of VIIQ217A and VIIQ217G to TF was comparable to that of wild-type VII to TF. All the variants were readily activated by factor Xa. Autoactivation in the presence of TF was efficient with VIIQ217E, slow with VIIQ217A, but undetected with VIIQ217G. Relative to wild-type VII added at the same concentration, VIIQ217E had no effect on the PT of normal plasma, whereas VIIQ217A slightly and VIIQ217G dramatically prolonged the clotting time in a dose-dependent manner. Activation of macromolecular substrates paralleled this functional inhibition. The k(cat)/K(M) values for factor X activation in the presence of TF were 2.4 for VIIaQ217E as compared to 1.9 (M(-)(1) s(-)(1) x 10(7)) for wild-type VIIa, 1.57 for VIIaQ217A, and 0.05 with VIIaQ217G. In comparison to wild-type VIIa, VIIaQ217E cleaved the chromogenic substrate S2765 (Z-D-Arg-Gly-Arg-pNA) with 10-fold higher k(cat). Analysis of the interactions with the inhibitors TFPI and antithrombin III demonstrated that VIIaQ217A but not VIIaQ217E or VIIaQ217G was inhibited less efficiently by TFPI either in the presence or in the absence of factor Xa. In contrast, VIIaQ217A association with antithrombin III in the presence of heparin was the fastest among the variants with a second-order rate constant of 2.31 (x10(3) M(-)(1) min(-)(1)), as compared to 0.47 and 1.47 for VIIaQ217E and wild-type VIIa, respectively. Our results demonstrate that residue Q(217) is important in regulating substrate and, more importantly, inhibitor recognition by VIIa.     

Immunocytochemical localization of S-100 beta beta protein in olfactory and supporting cells of lamb olfactory epithelium

By immunocytochemistry, we have identified two novel cell types, olfactory and supporting cells of lamb olfactory epithelium, expressing S-100 beta beta protein. S-100 immune reaction product was observed on ciliary and plasma membranes, on axonemes and in the cytoplasm adjacent to plasma membranes and to basal bodies of olfactory vesicles. A brief treatment of olfactory mucosae with Triton X-100 before fixation is necessary for detection of S-100 beta beta protein within olfactory vesicles. In the absence of such a treatment, the immune reaction product is restricted to ciliary and plasma membranes. On the other hand, irrespective of pre-treatment of olfactory mucosae, S-100 beta immune reaction product in supporting cells is restricted to microvillar and plasma membranes. The anti-S-100 beta antiserum used in these studies does not bind to basal cells of the olfactory epithelium or to cells of the olfactory glands, whereas it binds to Schwann cells of the olfactory nerve. An anti-S-100 alpha antiserum does not bind to cellular elements of the olfactory mucosa, Schwann cells, or axons of the olfactory nerve. The present data provide, for the first time, evidence for the presence of S-100 beta beta protein in mammalian neurons (olfactory cells).     

Reproductive cycle of trout and tench: effect of experimental variations of the temperature (author's transl)]

10 In the rainbow trout and the tench, at the time of spermatogonia divisions a rise in the plasma gonadotropin levels was observed. Just before or during the spawning period, E217 beta and plasma gonadotropin reach their maximum levels. 20 When the effects of raising the natural cycle temperature of the tench were studied (deltat + 3 degrees C and + 6 degrees C), in conditions where nycthemeral and seasonal rythmicity were maintained, a significant increase in fertility was observed with the increase in temperature (the first spawning period was earlier and there was a rise in the number of spawnings). This effect of temperature seems to act at the level of the hypothalamo-pituitary system.     

The development of a radioimmunoassay for formoterol

The development of a radioimmunoassay (RIA) for the beta 2-stimulant formoterol is described. The sensitivity of the method is 0.1 ng/ml in plasma and urine, when a 1-ml sample is used. The cross-reactivity of the antiserum with formoterol glucuronide was 30%. Since formoterol is metabolized extensively to formoterol glucuronide in rats, dogs and humans, extraction with ethyl ether prior to the radioimmunoassay was carried out. Satisfactory agreement was obtained for levels of formoterol in plasma and urine when they were determined by RIA and gas chromatography-mass spectrometry. The concentration of formoterol was determined in dog plasma and human urine after oral administration of formoterol fumarate to dogs (61 mcg/kg) and humans (40 mcg).     

Characterization of the transport properties of cloned rat multidrug resistance-associated protein 3 (MRP3).

We have previously cloned rat MRP3 as an inducible transporter in the liver (Hirohashi, T., Suzuki, H., Ito, K., Ogawa, K., Kume, K., Shimizu, T., and Sugiyama, Y. (1998) Mol. Pharmacol. 53, 1068-1075). In the present study, the function of rat MRP3 was investigated using membrane vesicles isolated from LLC-PK1 and HeLa cell population transfected with corresponding cDNA. The ATP-dependent uptake of both 17beta estradiol 17-beta-D-glucuronide ([3H]E217betaG) and glucuronide of [14C] 6-hydroxy-5, 7-dimethyl-2-methylamino-4-(3-pyridylmethyl) benzothiazole (E3040), but not that of [3H]leukotriene C4 and [3H]2, 4-dinitrophenyl-S-glutathione, was markedly stimulated by MRP3 transfection in both cell lines. The Km and Vmax values for the uptake of [3H]E217betaG were 67 +/- 14 microM and 415 +/- 73 pmol/min/mg of protein, respectively, for MRP3-expressing membrane vesicles and 3.0 +/- 0.7 microM and 3.4 +/- 0.4 pmol/min/mg of protein, respectively, for the endogenous transporter expressed on HeLa cells. [3H]E217betaG had also a similar Km value for MRP3 when LLC-PK1 cells were used as the host. All glucuronide conjugates examined (E3040 glucuronide, 4-methylumbelliferone glucuronide, and naphthyl glucuronide) and methotrexate inhibited MRP3-mediated [3H]E217betaG transport in LLC-PK1 cells. Moreover, [3H]methotrexate was transported via MRP3. The inhibitory effect of estrone sulfate, [3H]2,4-dinitrophenyl-S-glutathione, and [3H]leukotriene C4 was moderate or minimal, whereas N-acetyl-2,4-dinitrophenylcysteine had no effect on the uptake of [3H]E217betaG. The uptake of [3H]E217betaG was enhanced by E3040 sulfate and 4-methylumbelliferone sulfate. Thus we were able to demonstrate that several kinds of organic anions are transported via MRP3, although the substrate specificity of MRP3 differs from that of MRP1 and cMOAT/MRP2 in that glutathione conjugates are poor substrates for MRP3.     

Simultaneous radioimmunoassay of androstenedione, dehydroepiandrosterone and 11-beta-hydroxyandrostenedione in plasma

A simultaneous radioimmunoassay for delta 4-androstenedione (delta 4), dehydroepiandrosterone (DHA) and 11 beta-hydroxyandrostenedione (11 beta OH delta 4) in plasma is described. This involved preparing first an anti-11 beta-hydroxyandrostenedione-3-0-carboxymethyl oxime/BSA antiserum which binds both delta 4 and 11 beta OH delta 4, and an anti-dehydrosterone-7-0-carboxymethyl oxime/BSA antiserum. A chromatographic step using celite minicolumns separates these three steroids. The method was applied to the measurement of the plasma basal values of these three androgens in control subjects. Mean concentrations (ng/ml) of delta 4, DHA and 11 beta OH delta 4 were respecstively 1.35, 6.63 and 3.13 in males; 1.35, 6.65 and 2.59 in premenopausal females; 0.46, 1.53 and 1.38 in post-menopausal females, and 0.39, 0.73 and 1.78 in children 1--6 years of age. Dynamic tests were also carried out: ACTH stimulation was found to increase delta 4, DHA and 11 beta OH delta 4. Dexamethasone had a reverse effect causing a 50% diminution in delta 4 levels, a marked decrease in DHA levels, and a 90% decrease in 11 beta OH delta 4 levels. Metyrapone test was found to produce a 223% increase in delta 4 levels, a 196% increase in DHA levels, and a decrease of more than 90% in the 11 beta OH delta 4 levels. Estroprogestative drug treatment was accompanied by a decrease of not only delta 4, but also of DHA and 11 beta OH delta 4. Preliminary clinical results concerning these steroids show a parallel increase or decrease of delta 4 and 11 beta OH delta 4 in adrenal pathology. In ovarian hyperandrogeny, delta 4 is increased and 11 beta OH delta 4 is unchanged.     

Specific radioimmunoassay of estrone sulfate. Application to measurement in male plasma

We described a new, specific and easy to use radioimmunoassay (RIA) of estrone sulfate (E1S) in males. After synthesis of an E1S-6-(O-carboxymethyl) oxime hapten then coupling to BSA, we obtained a specific anti-E1S antiserum. Although the cross-reactivity of DHEAS with our anti-E1S antiserum was low (CR=0.002%), we confirmed the absolute necessity of separating plasma DHEAS from plasma E1S, before E1S RIA, because in plasma, DHEAS is present at levels 3-6000-fold higher than E1S, which generally is ignored. Thus, we elicited an easy separation of DHEAS from E1S, by a fast chromatography on in-house minicolumns. This new RIA, was applied to the determination of E1S plasma normal values in males. In 27 young men (<35 years), mean+/-S.D. were 1.97nmol/l+/-1.07nmol/l and in 63 untreated healthy aged men (>55 years), 1.80nmol/l+/-1.21nmol/l. No significant difference was seen between young and older subjects. The ranges of E1S plasma levels in these subjects were rather large and the ratios between the highest and the lowest E1S plasma levels were seven in the young group and 23.4 in the older group. No decrease of E1S plasma levels was observed with ages. Contrary to large interindividual E1S plasma level variations, the intraindividual variations have been found to be no significant. Correlations between E1S and unconjugated estrogens, E2 and E1 were 0.22 (P=0.016) and 0.51 (0.001), respectively.     

1H NMR studies of the mercuric ion binding protein MerP: Sequential assignment,secondary structure and global fold of oxidized MerP

Summary The oxidized form of the mercuric ion binding protein MerP has been studied by two-dimensional NMR. MerP, which is a periplasmic water-soluble protein with 72 amino acids, is involved in the detoxification of mercuric ions in bacteria with resistance against mercury. The mercuric ions in the periplasmic space are first scavenged by the MerP protein, then transported into the cytoplasm by the membrane-bound transport protein MerT, and finally reduced to elementary (nontoxic) mercury by the enzyme mercuric reductase. In this work, the ¹H NMR spectrum of oxidized MerP (closed disulfide bridge) has been assigned by using homonuclear 2D NMR techniques. The secondary structure and global fold have been inferred from the nuclear Overhauser effect (NOE) data. The secondary structure comprises four -strands and two -helices, in the order ₁₁₂₃₂₄. The protein folds into an antiparallel -sheet, ₂₃₁₄, with the two antiparallel helices on one side of the sheet. The folding topology is similar to that of acylphosphatase, the activation domain of porcine pancreatic procarboxypeptidase B, the DNA-binding domain of bovine papillomavirus-1 E2 and the RNA-binding domains of the U1 snRNP A and hnRNP C proteins. However, there is no structural similarity between MerP and other bacterial periplasmic binding proteins.     

Site-directed mutagenesis of an apolipoprotein E mutant, apo E5(Glu3----Lys) and its binding to low density lipoprotein receptors.

Apo E5(Glu3----Lys) is a naturally occurring apolipoprotein E (apo E) mutant found in patients with hyperlipoproteinemia and atherosclerosis. It has been shown to have a high affinity for low density lipoprotein (LDL) receptors. In this study, mutant apo E5 was produced by Chinese hamster ovary cells by means of an in vitro site-directed mutagenesis technique, and its LDL receptor binding activity was assessed. The apo E5 obtained from gene expression bound more readily to the LDL receptor than did plasma apo E3. The concentrations required for 50% competitive binding of 125I-labeled LDL to the LDL receptors were 58.9 ng/ml for plasma apo E3 and 25.7 ng/ml for the expressed apo E5. The expressed apo E5 displayed 229% normal binding. This result is highly consistent with that obtained with plasma apo E5, which showed 217% normal binding. Although the experimental apo E isoproteins contained more sialic acid than plasma apo E, the extent of sialylation had no effect on the receptor binding of apo E.     

Evidence for a rapid in vivo effect on estradiol-17 beta on prolactin secretion in ovariectomized rats.

Repeated intraarterial injections of synthetic thryrotropin releasing hormone (TRH, 1 microgram/rat) increased plasma prolactin levels 4 hours after a single subcutaneous injection of 10 micrograms estradiol-17 beta (E2-17 beta) in rats ovariectomized 1, 2 or 4 weeks and at 2 hours after E2-17 beta injection in rats ovariectomized for 6 weeks. The effect of TRH was still present at 24 but not 48 hours after estradiol treatment. TRH-induced increases in plasma prolactin were similar in groups of rats treated with 10 micrograms E2-17 beta (s.c.) or implanted with 0.5 cm Silastic capsules of crystalline E2-17 beta (s.c.) whereas smaller, yet significant, TRH-induced increases in plasma prolactin were observed in rats injected s.c. with 1.0 microgram E2-17 beta. Single intraarterial injections of TRH at 4 or 8 hours after E2-17 beta treatment induced increases in plasma prolactin similar in magnitude to those observed at the same times after E2-17 beta in rats given repeated TRH injections. No effect of TRH was observed in ovariectomized rats given sesame oil and E2-17 beta treatment did not influence plasma prolactin in rats given saline instead of TRH. Intraarterial administration of serotonin creatinine sulfate (5-HT, 10 mg/kg body weight) induced marked increases in plasma prolactin in rats ovariectomized for 4 weeks which were potentiated at 2 and 6 hours after E2-17 beta (10 micrograms) treatment. The data show that estradiol has a fairly rapid stimulatory effect on plasma levels of prolactin induced by two different secretagogues but the exact site and mechanism of action remain unresolved.     

Senile cataract-related accumulation of Lewis(x) glycolipid in human lens.

A glycosphingolipid that reacted positively to anti-stage-specific embryonic antigen-1 (SSEA-1) antiserum accumulated in human lens in association with aging and senile cataract formation. Since this antiserum recognizes Lewis(x) (Le(x)) structure, Gal beta 1-4(Fuc alpha 1-3)GlcNAc-, which is a typical tumor-associated and differentiation-related saccharide chain, the lens glycolipid was predicted to be a Lex antigen. The glycolipid purified from cataractous lens tissues was indeed a Lex glycolipid, Gal beta 1-4(Fuc alpha 1-3)GlcNAc beta 1-3Gal beta 1- 4Glc beta 1-1 ceramide. Enhanced expression of the Lex glycolipid may affect the organization of lens plasma membranes through Le(x)-Le(x) interactions, as suggested for compaction in mouse preimplantation embryos and embryonic teratocarcinomas, resulting in lens opacification, namely cataract.     

Equol: a contributor to enigmatic immunoassay measurements of estrogen

The efficacy of radioimmunoassay (RIA) for the measurement of estradiol-17 beta (E2) in murine plasma was investigated. When Sephadex LH-20 or celite column chromatography was used to separate E2 from estrone (E1) and other cross-reacting compounds, the results were erratic if small volumes of mouse plasma were resolved. Assay of a diethyl ether extract of plasma (500 microL) was the most practical method for estimating the concentration of estradiol-17 beta in mice. This method was used to determine the pattern of estrogen secretion during the estrous cycle, on the day of implantation and during pregnancy. No convincing change in estrogen secretion was observed in the diestrous/proestrous mouse. By comparison, estrogen levels were elevated during pregnancy. Taken together, these results implied that cross-reactive components in plasma masked low levels of endogenous estrogen. Further evaluation of mouse plasma and urine using a co-chromatography technique to examine estrogen elution from a reverse-phase HPLC system followed by GC/MS analysis indicated the presence of equol [7-hydroxy-3-(4-hydroxyphenyl)chroman], a phytoestrogen metabolite with a ring structure similar to estradiol-17 beta. Equol and possibly other cross-reactive components of plasma may account for the apparent lack of increased estrogen secretion during the mouse estrous cycle and on the day of implantation as determined by the radioimmunoassay of ether extracts of plasma.     

Immunological identification of G protein alpha- and beta-subunits in tail membranes of bovine spermatozoa.

Heterotrimeric G proteins are believed to play important roles as signal transducing components in various mammalian sperm functions. To assess the distribution of G proteins in bovine sperm tails, we purified membranes by hypoosmotic swelling of bovine spermatozoa followed by disruption of plasma membranes in a homogenizer and various centrifugation steps. Electron microscopy revealed highly purified membranes of bovine sperm tails. Subsequently, antisera against synthetic peptides were used to identify G proteins in immunoblots. An antiserum directed against the C-terminal decapeptide of Gi3 and detecting all known pertussis toxin-sensitive alpha-subunits, reacted specifically with a 40-kDa protein. In contrast, various other specific peptide antisera against alpha-subunits did not detect any G protein in enriched tail membranes. An antiserum recognizing the beta 2-subunit of G proteins and an antiserum reacting with both beta 1- and beta 2-subunits identified a 35-kDa protein in sperm tail membranes. In contrast, antisera against the 36-kDa beta 1-subunit did not detect any relevant proteins in the membrane fraction. Neither G protein alpha-subunits nor G protein beta-subunits were found in the cytosol. Our results suggest that G proteins in membranes of tails of bovine spermatozoa most likely belong to a novel subtype of G protein alpha-subunits, whereas the putative beta-subunit could be identified as a beta 2-subunit.     

The effect of anti-beta 43-47 Fab fragments on expression of the polymerization sites in the E domain of fibrinogen

In order to assess the significance of the aminoterminal residues of the B beta chain in expression of polymerization sites of the E domain, we have prepared polyclonal antiserum against a synthetic peptide corresponding to beta 43-47 of human fibrinogen. Affinity purified immunoglobulin IgG and Fab prepared from these antibodies reacted strongly and specifically with the synthetic pentapeptide and intact fibrinogen molecule. This specificity was determined both by radioimmunoassay and Western blot analysis of fibrinogen and its plasmic fragments and described in our previous paper (Cierniewski et al., 1986, Biochim. Biophys. Acta, 884, 594-597). Immunochemically purified anti beta 43-47 antibodies and their Fab fragments were strong inhibitors of the fibrin monomer polymerization. Our results imply that amino acid sequence beta 43-47 recognized by these antibodies may be in a close vicinity to the contact sites of the E domain.     

Ethynylestradiol-17 beta D-ring glucuronide conjugates are potent cholestatic agents in the rat

17 alpha-Ethynylestradiol-17 beta (beta-D-glucuronide) [EE217 beta (beta G)], a metabolite of 17 alpha-ethynylestradiol (EE2) identified in urine of women taking EE2 in oral contraceptives, and its synthetic anomer, 17 alpha-ethynylestradiol-17 beta (alpha-D-glucuronide), [EE217 beta (alpha G)], were administered intravenously to female rats in order to determine their effects on bile flow. Both agents induced an immediate, profound and dose-dependent decrease in bile flow which returned to control levels within 1-8 hr. The logarithm of the dose vs the cholestatic response curves for the two anomers were not parallel. EE217 beta (alpha G) was significantly more potent than EE217 beta (beta G) such that the doses inhibiting bile flow by 50% were 1.25 and 11 mumol/kg for the alpha-and beta-anomer respectively.     

Human F1-ATPase: molecular cloning of cDNA for the beta subunit

F1-ATPase is the major enzyme for ATP synthesis, and its beta subunit is the catalytic site. To date, no full-length cDNA for the eukaryotic F1 gene has been reported. Human F1 was studied because of its importance in medicine and cell biology. Here we report molecular cloning of a full-length cDNA for the human F1 beta subunit and purification of the human F1 beta subunit. The HeLa cell cDNA library constructed in an expression vector gamma gt11 was screened with antiserum against the yeast F1 beta subunit. One of the positive phage DNAs containing the human F1 beta gene and its flanking regions (1.8 kilobase pairs) was sequenced by the dideoxy chain termination method. The open reading frame started from a putative signal presequence, which was rich in both serine and arginine. There was a homologous segment in the signal presequence of human ornithine transcarbamoylase and that of F1 beta. The precursor of F1 beta was expressed in E. coli harboring a plasmid which had been constructed with T5 promotor and the F1 beta cDNA. Both the precursor and mature form of F1 beta were detected in HeLa cells in a pulse-chase experiment. The amino acid sequence of 480 residues (51,568.3 daltons) following the presequence was highly homologous with that of mature beef heart F1 beta (97.5%) and E. coli F1 beta (71.7%), but the codon usage in the human gene was very different from those of reported genes coding for F1 beta of other species.