Evidence of sex hormone binding globulin binding sites in the medial preoptic area and hypothalamus.

We have demonstrated a high density of both radiolabeled progesterone and estradiol conjugated to bovine serum albumin binding sites in the medial preoptic area and hypothalamus. Infusions of sex hormone binding globulin into the medial preoptic area of rats increased their female sexual receptivity similarly to the effect of estradiol conjugated to bovine serum albumin, suggesting sex hormone binding globulin acts at binding sites for estradiol conjugated to bovine serum albumin. In this study sex hormone binding globulin was used to displace radiolabeled progesterone conjugated to bovine serum albumin from plasma membrane fractions from the medial preoptic area-anterior hypothalamus and medial basal hypothalamus of ovariectomized rats injected with either 5 microg estradiol benzoate or sesame oil vehicle. We found that sex hormone binding displaced radiolabeled progesterone conjugated to bovine serum albumin in both areas and that in vivo estradiol treatment greatly increased the relative displacement by sex hormone binding globulin in the medial preoptic area-anterior hypothalamus. We interpret these data as indicating the presence of sex hormone binding globulin receptors in brain plasma membranes and further suggest that endogenous steroid conditions may alter these receptors.     

Acute reduction of serum leptin level by dietary conjugated linoleic acid in Sprague-Dawley rats

The purpose of this study was to clarify the effect of conjugated linoleic acid on lipid accumulation in adipose tissue. Sprague-Dawley rats were fed a diet containing 2% conjugated linoleic acid for 1, 3, 6, and 12 weeks. In rats fed 2% conjugated linoleic acid, the weight of perirenal white adipose tissue was comparable with that of rats fed a conjugated linoleic acid-free diet. For fatty acid composition of perirenal white adipose tissue, both 16:1/16:0 and 18:1/18:0 ratios were significantly lower in the conjugated linoleic acid-fed group than the control group. Although there was no remarkable difference in serum triglyceride, total cholesterol, and phospholipid levels between dietary groups, serum leptin level was significantly lower than the control group, and lipid content in the perirenal white adipose tissue exerted a tendency toward low compared to the control value at 1-week feeding. On the other hand, leptin level in perirenal white adipose tissue was significantly lower in the conjugated linoleic acid-fed group than the control group at 12-week feeding. In conclusion, these observations suggest dietary conjugated linoleic acid is an acute reducer of serum leptin level. This may afford an explanation of the mechanism of anti-obesity effect in conjugated linoleic acid.     

Estrogen metabolism in the Asian elephant (Elephas maximus)

Estradiol-17β metabolism was studied in two female Asian elephants (Elephas maximus). In an initial study, 500 μCi of tritiated estradiol-17β was injected iv into a single animal, and 0, 30 and 60 min serum samples were collected as well as all excreted urine and feces for 24 hr. In a second study, 1.5 mg unlabeled estradiol-17β was injected iv into a second animal and 0, 5, 15, 30, and 60 min serum samples and a 30 min urine sample were collected postinjection. Analyses of samples from both studies demonstrated a rapid conversion of free estradiol to conjugated forms in the serum. The first (5 min) serum sample following the injection of unlabelled estradiol contained unconjugated estradiol: conjugated estradiol: conjugated estrone at a ratio of 60: 29: 10, respectively, and at 30 min a ratio of 33: 43: 24. The urinary estrogen metabolites were in the conjugated form with an estradiol: estrone ratio of 60: 40. No radiolabeled estrogen was found in the fecal samples during the 24 hr following administration of the radiolabeled estradiol. These data indicate a rapid clearance of circulating free estradiol in the elephant, with a major metabolite in the serum and urine being estradiol conjugate. © 1992 Wiley-Liss, Inc.     

Kringle 5 peptide-albumin conjugates with anti-migratory activity

Three peptide fragments of the kringle 5 region of plasminogen and their respective N- and C-terminus maleimido derivatives conjugated to Cys34 of human serum albumin were evaluated in vitro using a human umbilical vein endothelial cell (HUVEC) migration assay and a human plasma stability assay. The N-terminus maleimido derivative of the 64 to 74 segment of kringle 5 conjugated to human serum albumin possessed remarkable anti-migratory activity.     

Chronic cigarette smoking alters circulating sex hormones and neuroactive steroids in premenopausal women

Chronic smoking alters the circulating levels of sex hormones and possibly also the neuroactive steroids. However, the data available is limited. Therefore, a broad spectrum of free and conjugated steroids and related substances was quantified by GC-MS and RIA in premenopausal smokers and in age-matched (38.9+/-7.3 years of age) non-smokers in the follicular (FP) and luteal phases (LP) of menstrual cycle (10 non-smokers and 10 smokers, in the FP, and 10 non-smokers and 8 smokers in the LP). Smokers in both phases of the menstrual cycle showed higher levels of conjugated 17-hydroxypregnenolone, 5alpha-dihydroprogesterone, conjugated isopregnanolone, conjugated 5alpha-pregnane-3beta,20alpha-diol, conjugated androstenediol, androstenedione, testosterone, free testosterone, conjugated 5alpha-androstane-3alpha/beta,17beta-diols, and higher free testosterone index. In the FP, the smokers exhibited higher levels of conjugated pregnenolone, progesterone, conjugated pregnanolone, lutropin, and a higher lutropin/follitropin ratio, but lower levels of cortisol, allopregnanolone, and pregnanolone. In the LP, the smokers exhibited higher levels of free and conjugated 20alpha-dihydropregnenolone, free and conjugated dehydroepiandrosterone, free androstenediol, 5alpha-dihydrotestosterone, free and conjugated androsterone, free and conjugated epiandrosterone, free and conjugated etiocholanolone, 7alpha/beta-hydroxy-dehydroepiandrosterone isomers, and follitropin but lower levels of estradiol and sex hormone binding globulin (SHBG) and lower values of the lutropin/follitropin ratio. In conclusion, chronic cigarette smoking augments serum androgens and their 5alpha/beta-reduced metabolites (including GABAergic substances) but suppresses the levels of estradiol in the LP and SHBG and may induce hyperandrogenism in female smokers. The female smokers had pronouncedly increased serum progestogens but paradoxically suppressed levels of their GABA-ergic metabolites. Further investigation is needed concerning these effects.     

Synthesis and immunomodulating tumor control properties of albumin covalently connected to trehalose

Summary Proteins conjugated to a trehalose derivative are described. These include human serum albumin and bovine serum albumin covalently attached through appropriate molecular linkages to the 6- and 6,6-positions of trehalose. The immunomodulating tumor control activities of these conjugated proteins has been assayed in several test systems.     

The influence of conjugated estrogens in radioimmunoassays using different antibodies against estradiol-17β

Estradiol-17β 17-D-glucoronide and estrone-3-sulphate were found to give minor crossreaction in radio-immunoassay systems for estradiol-17β using two different types of antibodies. The antibodies used were prepared in sheep against estradiol-17β 17-succinyl-bovine serum albumin and 6-ketoestradiol-17β 6-(0-carboxymethyl)oxime-bovine serum albumin. Only minute quantities of conjugated estrogens were extracted from plasma by diethyl ether (<0.5 %). These two factors limit the interference of conjugated estrogens.     

An indirect fluoresent antibody staining procedure for detection of Vibrio cholerae serovar 01 cells in aquatic environmental samples

An immunofluorescence method for detection of Vibrio cholerae serovar 01 in aquatic environmental samples and enrichment broths is described. Antiserum specific for the 01 somatic antigen was produced in rabbits and used in an indirect fluorescent antibody method incorporating fluoresceinisothiocyanate conjugated anti-rabbit globulin goat serum, and rhodamine isothiocynate conjugated bovine serum albumin as background stain. Comparisons of the immunofluorescent procedure and conventional culture methods for isolation of V. cholerae 01 showed that detection occurred significantly more frequently with the fluorescent antibody system.     

Detection of IgA defects in blood donors using an enzyme immunoassay

An enzyme immunoassay is presented for screening blood donors for IgA-defects using horseradish peroxidase conjugated IgA. Fife of about 4,700 screened donors was found to be IgA-deficient. With another EIA using horseradish peroxidase conjugated monoclonal anti-IgA and IgA coated microplates the IgA-concentration in serum of these selected donors was determined.     

Comparative Trial of P1496, a New Non-steroidal Oestrogen Analogue

The results are reported of a preliminary trial of P1496, a new non-steroidal oestrogen analogue, compared with a conjugated equine oestrogen and a placebo. The oestrogenicity of both substances was well substantiated by vaginal epithelial maturation indices. P1496 was superior to conjugated equine oestrogen in producing a significant reduction of plasma calcium levels and a possible reduction in serum cholesterol. Conjugated oestrogen caused slightly more nausea than P1496 but there were no notable side effects from either drug. P1496 is considered to be at least as effective an oestrogenic substance as conjugated oestrogen and worthy of further therapeutic evaluation.     

Visualization of surface antigens on yeast protoplasts by gold or ferritin markers

Surface antigens of Saccharomyces cerevisiae protoplast were visualized immunocytochemically with protein A-gold or ferritin conjugated antibodies. Ferritin particles were densely distributed at the surface of yeast protoplast after treatment with rabbit anti-protoplast serum and ferritin conjugated antibody against rabbit IgM. Control experiments demonstrated that the binding of these ferritin markers was specific for surface antigens. The composition of these surface antigens, however, remain to be investigated.     

The dynamics of ubiquitin pools within cultured human lung fibroblasts

The dynamics of ubiquitin pools within the cultured human lung fibroblast line IMR-90 were examined using solid phase immunochemical methods to quantitate free and conjugated polypeptide. Fetal calf serum was found to contain a nondialyzable factor that induced a transient accumulation of ubiquitin. During the induction, free and conjugated ubiquitin pools changed in concert so that the fraction conjugated remained constant. The induction of ubiquitin by the serum factor resulted from an enhanced rate of protein synthesis. Within experimental error no change in the first order rate constant for intracellular ubiquitin degradation was observed. Pulse-chase studies revealed ubiquitin to turn over with a half-life of 28-31 h in conditioned and freshly fed cultures. Withdrawal of serum from cultures led to a rapid decline in total ubiquitin during which the fractional level of conjugation remained constant. The accelerated ubiquitin turnover following removal of serum likely involves lysosomal autophagy since 10 mM NH4Cl led to an accumulation of the polypeptide. Since no similar effect of the lysosomotropic compound was observed in conditioned or freshly fed cultures, nonlysosomal processes are probably responsible for ubiquitin turnover under nutritional balance. The dynamics of these intracellular pools suggests that the ubiquitin ligation system is subject to regulatory constraints not previously suspected. The short half-life for ubiquitin is consistent with the apparent ability of cells to alter ubiquitin levels in response to external stimuli and stress.     

A convenient, unified scheme for the differential extraction of conjugated and unconjugated serum C19 steroids on Sep-Pak C18-cartridges

In order to conveniently and rapidly isolate by group both conjugated and unconjugated serum androgens, a scheme has been devised for their differential extraction from commercially available, disposable octadecylsilane cartridges (Sep-Pak C18). Using added radioactive steroid standards and detection of endogenous serum steroids by group-specific enzymatic assays, the quantitative recovery of steroid glucuronides and sulfates in the 47% methanol fraction and of unconjugated steroids in the 100% methanol fraction was observed. Maximum recovery of serum protein-bound steroids (e.g. testosterone) was achieved with serum denatured by urea and heat. In order to separate glucuronides from sulfates, sequential hydrolysis of the conjugated fraction (47% methanol) by enzymatic hydrolysis and then organic solvolysis as well as an additional Sep-Pak cartridge extraction step was required. Groups of extracted steroids may be further separated and assayed by any appropriate method(s). An application is given which employs HPLC and an enzymatic assay for 17 beta-hydroxy- and 17-oxo-steroids to provide separate profiles of unconjugated, glucuronidated, and sulfated androgens in human, male serum.     

Prolonged in vivo residence times of antibody fragments associated with albumin

Antibody fragments can be expressed at a high level in microbial systems, but they may have limited therapeutic value because they are rapidly eliminated from the body. We demonstrate here that site-specific conjugation or binding of bacterially derived Fab' to the long-lived protein serum albumin allows full retention of the antibody's binding characteristics while imparting the albumin's longevity in vivo. In rats the area under the curve for Fab' conjugated to rat serum albumin was 17-fold greater than for the control of Fab' conjugated to cysteine. Again, a bispecific F(ab')(2) with specificity for rat serum albumin showed an area under the curve about 8-fold greater than did a F(ab')(2) without specificity to albumin. Genetic fusions of scFv to albumin were similarly long-lived and could be expressed in yeast to provide the basis of a cost-effective production system.     

Human serum albumin interaction,in silico and anticancer evaluation of Pine-Gold nanoparticles

Unique characteristics displayed by phytoconstituent conjugated nanoparticles and their crucial interactions with proteins serve to develop nanoparticle-bio-interface platform. Gold nanoparticles of 16 nm in size were generated using aqueous extracts of pine bark and further conjugated to human serum albumin. The gold nanoparticles-protein complex was characterized by surface plasmon resonance, UV–vis and emission spectroscopy techniques. Further, it was characterized for surface morphology and elemental composition, crystallographic quality, nanoparticles size, shape, stability, structural determination and the identification of capping agent. Moreover, the interaction of gold nanoparticles with human serum albumin was investigated using conventional spectroscopy techniques. Fluorescence quenching and absorption studies demonstrated an effective binding of human serum albumin with oleamide capped gold nanoparticles. The molecular docking study showed a binding affinity of -6.1 kcal/mol whereas the molecular dynamics simulation indicated that the binding of oleamide to human serum albumin. A biological evaluation of pine bark extract-gold nanoparticles showed cytotoxicity with increased cell mortality in lung cancer cells and minimal toxicity on non-cancerous human embryonic kidney cells, respectively.     

Anti-bilirubin monoclonal antibody. II. Enzyme-linked immunosorbent assay for bilirubin fractions by combination of two monoclonal antibodies

Among 16 monoclonal antibodies raised against covalently coupled bilirubin-bovine serum albumin, we selected two antibodies: one (designated 24G7) reacted with unconjugated and conjugated bilirubin and the other (designated 25H17) reacted only with unconjugated bilirubin. Combination of these two antibodies enabled us to determine extremely low concentrations of unconjugated and conjugated bilirubin independently by enzyme-linked immunosorbent assay (ELISA). In the assay, samples were incubated with each anti-bilirubin IgG, and then free remaining IgG was allowed to bind to the immunotiter plates coated with bilirubin-bovine serum albumin. The bound fraction of the IgG was visualized with horseradish peroxidase-conjugated rabbit anti-mouse IgG and substrate. Bilirubin concentration was determined from the absorbance at 425 nm. In this system, we could measure 10(-7)-10(-5) M unconjugated and conjugated bilirubin in samples, which is 100-fold more sensitive than Micha?lsson's diazocoupling method. The assay results gave a good correlation coefficient (0.86) compared with those determined with high performance liquid chromatography.     

RGD Peptide–Albumin Conjugate for Endothelization of Electrospun Materials

Russian Journal of Bioorganic Chemistry - To improve the endothelization of 3D matrices produced by electrospinning we propose adding cyclo(RGDfC) peptide conjugated to human serum albumin (HSA)...     

Determination of estrogens by radioimmunoassay with antibodies to estrogen-C6-conjugates. II. Synthesis of estradiol-17alpha-6-albumin conjugate

The 6-carboxymethoxime of 6-oxo-estradiol-17alpha was prepared and conjugated to bovine serum albumin by the mixed anhydride technique. After purification, 31 6-oxo-estradiol residues were covalently bound to 1 molecule albumin.     

Conjugate of bovine serum albumin with nicotine

The preparation and characterization of a nicotine-albumin conjugate are reported. The nicotine derivative, 6-(p-aminobenzamido)nicotine, which was coupled to bovine serum albumin(BSA), was synthesized by the following sequence; $\text{1}$-nicotine→6-aminonicotine→6-(p-nitrobenzamido)nicotine→6-(p-aminobenzamido)nicotine. Thirty molecules of conjugated nicotine per molecule of BSA were distinguished by the determination of 6-aminonicotine after hydrolysis. The distribution of the conjugated nicotine on BSA was examined by the stoichiometry of the composed amino acids before and after conjugation, and also by the electrophoretic behavior of the conjugated BSA on a polyacrylamide gel.     

Identification of a diene conjugated component of human lipid as octadeca-9,11-dienoic acid

The predominant diene conjugated acyl residue in triacylglycerols, cholesteryl esters and phospholipids in human serum was identified by high performance liquid chromatography and capillary gas chromatography-mass spectrometry. It is an octadeca -9,11-dienoic acid.