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Resistance of alpha-globulin from Sesamum indicum L. to proteases in relationship to its structure.

alpha-Globulin, the high-molecular-weight protein fraction from Sesamum indicum L., was hydrolyzed to low-molecular-weight protein and peptides by pepsin, while its resistance to hydrolysis by group-specific enzymes, trypsin or alpha-chymotrypsin, was very high. The protein showed definite structural changes after proteolysis, especially after peptic hydrolysis, as evidenced from various biophysical data. The sedimentation velocity pattern of alpha-globulin hydrolyzed by trypsin or alpha-chymotrypsin indicated reduction in the percentage of 11S component, while the pepsin-hydrolyzed sample was devoid of any 11S component, indicating the absence of a native protein molecule. The fluorescence emission spectra of the various hydrolyzed alpha-globulin showed a red shift in the fluorescence emission maximum. The red shift was maximum with alpha-globulin hydrolyzed by pepsin and minimum with the trypsin-hydrolyzed sample. The far-ultraviolet-circular dichroic measurements indicated that most of the ordered structure of alpha-globulin was absent after pepsin hydrolysis, while after trypsin and chymotrypsin hydrolysis conformational changes were less.

Alpha-Globulins↗

Therapeutic efficacy of human anti-tetanic globulin--a clinical study of 100 cases.

The present study was performed to evaluate clinically the therapeutic efficacy of human anti-tetanic globulin in established cases of tetanus. The study was conducted in 100 patients in India with tetanus receiving treatment at P.B.M. Hospital, Bikaner. The human anti-tetanic globulin was given in addition to equine anti-tetanic serum, to all those patients who could afford to purchase the same, irrespective of age, sex or the severity of the disease, during the period of study. Except for the administration of human anti-tetanic globulin, the management of all the patients was identical. The study revealed that human anti-tetanic globulin is effective for patients with a short period of onset. Patients having prolonged spasms at frequent intervals were also the beneficiaries, to some extent. The hospitalization period of all the patients receiving human anti-tetanic globulin was shortened.

Adolescent↗

High-resolution FISH mapping of the rat alpha2u-globulin multigene family.

The rat alpha2u-globulins are a group of similar proteins that are encoded by a family of approximately 20 genes located a single locus of < or =880 kbp on Chromosome (Chr) 5q. Individual members of this gene family demonstrate complex tissue, hormonal, and developmental expression patterns despite a high degree of sequence similarity among the members and consequently provide an interesting system for studying the evolution of differential gene expression. Hybridization analysis indicated that gene classes, similar to those identified at the homologous MUP locus in the mouse, do not exist within the rat alpha2u-globulin locus. Furthermore, cross-hybridization analysis revealed the presence of conserved sequences in the 5' and 3' regions flanking the alpha2u-globulin genes, some of which were present in an inverted orientation. We have used high-resolution fiber FISH to examine the structural organization of the alpha2u-globulin locus, and found the genes to be arranged as an array of both direct and inverted repeats. The organization of the rat alpha2u-globulin genes differs from the MUP genes and suggests different evolutionary events have reorganized these homologous sets of genes.

Alpha-Globulins↗

Divergent effects of two low-dose oral contraceptives on sex hormone-binding globulin and free testosterone.

The effect of a triphasic combination of ethinyl estradiol and levonorgestrel on the serum concentrations of total testosterone, free testosterone, and sex hormone-binding globulin as measured directly by radioimmunoassay and on the binding capacity of sex hormone-binding globulin was compared with that of a preparation containing ethinyl estradiol and desogestrel. Blood samples were taken on days 6, 11, 21, and 28 of a control cycle, the third cycle of treatment with either ethinyl estradiol-levonorgestrel or ethinyl estradiol-desogestrel (11 volunteers each), the third cycle of a 3-month washout period, and the third treatment cycle after crossover change of the preparations. There was a significant reduction in total testosterone by 16% during treatment with both preparations. Ethinyl estradiol-desogestrel increased the concentration (+175%) and binding capacity (+330%) of sex hormone-binding globulin to a much greater extent than with ethinyl estradiol-levonorgestrel (+92% and +160%). Contrary to this, a significant suppression of non-protein-bound testosterone by 35% was found during treatment with both oral contraceptives. The results demonstrate that an excessive elevation of the levels of sex hormone-binding globulin above the normal range does not cause a corresponding suppression of free testosterone. It is assumed that the decrease in the apparent binding affinity of high sex hormone-binding globulin concentrations to testosterone may be due to protein-protein interactions.

Adult↗

Serum pharmacokinetics of orally administered desogestrel and binding of contraceptive progestogens to sex hormone-binding globulin.

Serum levels of 3-ketodesogestrel and ethinyl estradiol were analyzed by radioimmunoassay in a balanced crossover study with two tablet formulations containing desogestrel (0.150 mg) and ethinyl estradiol (0.030 mg) in 25 women under steady-state conditions after 21 days of treatment. The pharmacokinetic properties of desogestrel were characterized by the following parameters: (1) maximum serum concentration, (2) time to maximum serum concentration, (3) total area under the serum concentration versus time curve, and (4) serum half-life of elimination. The interindividual variation in these parameters was comparable with that observed with other contraceptive combinations containing ethinyl estradiol and norethisterone, levonorgestrel, or gestodene. The serum distribution of contraceptive progestogens is known to be determined by their affinity to sex hormone-binding globulin and the concentration of sex hormone-binding globulin. We analyzed the structural features that determine binding to sex hormone-binding globulin. The 18-methyl group increased and the 11-methylene group weakened the binding to sex hormone-binding globulin. The double bond at C-15 reinforced the binding only when combined with an 18-methyl group. Therefore, the binding of levonorgestrel (the 18-methyl derivative of norethisterone) and gestodene (the delta-15,18 methyl derivative of norethisterone) to sex hormone-binding globulin was much stronger than that of 3-keto-desogestrel and norethisterone.

Administration, Oral↗

Gamma globulin-derived standards for the determination of molecular weights, transfer, and immunodetection efficiencies in protein blotting procedures.

Molecular weight markers which are detectable using labeled antispecies antibodies or labeled Protein A have been prepared for use as standards on protein blots. The standards were prepared by the controlled reduction followed by subsequent alkylation of gamma globulin. Separate sets of standards were prepared using gamma globulins derived from human, mouse, rabbit, and sheep species. Standards were also prepared using monoclonal-derived gamma globulins from human myeloma fluid and mouse ascites fluid. Standards produced from monoclonal-derived gamma globulins produced very sharp bands on sodium dodecyl sulfate-polyacrylamide gels and proved to be excellent standards for this technique alone. However, the markers were uniquely suitable for use as standards in protein blotting procedures because their detection was achieved by the procedure used to detect the transferred antigen(s). The detection of immunoglobulin G (IgG)-derived standards on protein blots from all the species listed above was demonstrated using appropriate horseradish peroxidase (HRP)-conjugated antispecies antibodies. The use of other detection systems (biotin-labeled antibody and subsequent detection with HRP-steptavidin, HRP-Protein A) was also validated with human IgG-derived standards. Furthermore, the standards were shown to be suitable for use on both nitrocellulose and cationized nylon-based supports and could be used when adjacent samples were run under reducing conditions. Hence the gamma globulin-derived standards serve as both a control to check the adequacy of transfer and immunodetection systems and as markers which enable the molecular weights of detected antigens to be calculated.

Animals↗

Plasma vitamin D-binding globulin in vitamin D deficiency, pregnancy and chronic liver disease.

Plasma concentrations of vitamin D-binding globulin were measured by radial immunodiffusion in healthy subjects, pregnancy, and during oestrogen therapy. Subjects with disorders of vitamin D metabolism (dietary deficiency, malabsorption, anticonvulsant therapy, chronic liver disease) were also studied. Neither sex nor age influenced the plasma vitamin D-binding globulin concentration in healthy subjects, but there was a significant increase in concentration during pregnancy and oestrogen therapy. Elevated levels were found in vitamin D deficient elderly but not younger subjects, while levels in subjects with chronic liver disease were significantly reduced. Normal levels of vitamin D-binding globulin were present in hypervitaminosis D and no vitamin D-binding globulin was detected in human milk. No correlation was observed between plasma 25-hydroxycholecalciferol levels and plasma vitamin D-binding globulin concentrations.

Adult↗

Effects of K+ channel openers on the vascular actions of human gamma globulin.

The aim of this study was to determine if the stimulatory action of human gamma globulin on the spontaneous activity of the rat mesenteric portal vein is due to decreased K+ conductance. Glibenclamide potentiated the action of human gamma-globulin on the portal vein by 45% and on its own had a concentration- and time-dependent biphasic (increase followed by a decrease) effect on the spontaneous activity of the portal vein. Diazoxide and pinacidil both inhibited the action of human gamma-globulin on the rat mesenteric portal vein. Levcromakalim (BRL 38227) potentiated the stimulatory action of human gamma-globulin on the integrated force of the spontaneous contractions of the rat mesenteric portal vein by 40% and 49% at concentrations of 0.5 and 5 microM, respectively. These studies suggest that human gamma-globulin can act by directly modulating a K+ channel.

Animals↗

Acute nephrotoxicity of a carcinogenic iron chelate. Selective inhibition of a proteolytic conversion of alpha 2U-globulin to the kidney fatty acid-binding protein.

The mechanism of acute nephrotoxicity of an iron chelate in vivo has been investigated. Administration of a renal carcinogen ferric nitrilotriacetate (Fe-NTA) (15 mg Fe/kg body weight, intraperitoneally) led to selective loss of a renal protein with an apparent molecular mass of 17 kDa. Analysis of the 17 kDa protein by NH2-terminal sequence demonstrated its identity over 16 NH2-terminal residues as a kidney fatty acid-binding protein (k-FABP) that is a proteolytically modified form of alpha 2U-globulin, a major urinary protein of adult male rats. An immunochemical study using anti-alpha 2U-globulin polyclonal antibodies confirmed that a single injection of Fe-NTA led to a decrease in k-FABP levels. However, a 19-kDa protein identical to the alpha 2U-globulin progressively appeared in the kidney, suggesting that the proteolytic processing of alpha 2U-globulin in the renal proximal tubules was suppressed by the treatment with Fe-NTA. By monitoring k-FABP and its precursor alpha 2U-globulin, it was determined that repeated exposure to Fe-NTA caused suppression of both proteolytic and endocytotic activity of the kidney. The implications of these data in relation to the nephrotoxicity of Fe-NTA are discussed.

Alpha-Globulins↗

Treatment of pneumococcal postsplenectomy sepsis in the rat with human gamma-globulin.

A rat model was used to evaluate the possible effect on experimental postsplenectomy sepsis of a human gamma-globulin preparation for intravenous use (Sandoglobulin). Sixty splenectomized male Sprague-Dawley rats were given 3 X 10(3) Streptococcus pneumoniae type 1 intravenously. Twelve of the animals received no treatment and all died, in contrast to 12 sham-operated controls which all survived the challenge. The remaining splenectomized rats were divided into four groups, each consisting of 12 animals. One group was given 120 mg human gamma-globulin twice intraperitoneally (0.3 g/kg body wt), at 18 and 42 hr, after challenge; 10 of the 12 survived, in contrast to none of the 12 in the second group receiving 120 mg human albumin instead of gamma-globulin (P = 0.00003). When the injections were delayed to 24 and 48 hr, 9/12 gamma-globulin-treated animals still survived, in contrast to 0/12 in the albumin group. These findings point to new possibilities for treatment and perhaps prevention of overwhelming postsplenectomy sepsis by administration of high doses of gamma-globulins.

Albumins↗

Effects of a major androgen-dependent urinary protein, alpha 2u-globulin on the pituitary-gonadal axis and hypothalamic monoamines in adult male mice.

The purpose of the present study was to evaluate the effects of alpha-2u-globulin, a sex-dependent male rat urinary protein on pituitary-gonadal functions and hypothalamic monoamine contents in male mice. Adult male mice, maintained under standardized laboratory conditions (L:D, 14:10) were injected subcutaneously with alpha-2u-globulin at a dose of 1 mg/animal/day or with vehicle daily for 14 days and killed 16 h after the last injection. Plasma levels of luteinizing hormone (LH), follicle-stimulating hormone (FSH), testosterone (T) and testicular levels of T were measured by radioimmunoassays. The concentrations of norepinephrine (NE), dopamine (DA) and serotonin (5-HT) in medial basal hypothalamus (MBH) and anterior hypothalamus (AH) were measured by high performance liquid chromatography. Administration of alpha-2u-globulin led to a significant increase in plasma FSH and LH levels (P less than 0.05) as well as in plasma and testicular T levels (P less than 0.025). In the MBH of alpha-2u-globulin treated mice, there were significant elevations of NE (P less than 0.025), DA (P less than 0.01) and 5-HT (P less than 0.025) contents. In the AH, both DA (P less than 0.025) and 5-HT (P less than 0.01) contents were decreased while NE content remained unaltered. These results indicate that administration of alpha-2u-globulin can lead to a significant stimulation of pituitary-testicular axis and that this effect may be mediated through alteration of hypothalamic monoamines.

Alpha-Globulins↗

Rice seed globulin: a protein similar to wheat seed glutenin.

The globulin of the seed endosperms of rice has an isoelectric point of 6.4 and a M(r) of 26,000. There is one intra-disulphide bond, but no N-linked oligosaccharide chain. The amino acid sequence of the N-terminal and internal regions of the globulin was determined and found to be homologous with that of glutenin, the storage protein in the seed endosperms of wheat. Rice globulin and wheat glutenin were rich in glycine, and glutamic acid or glutamine, and in addition, wheat glutenin cross-reacted with antibody raised against rice globulin. These results suggest that rice seed globulin represents a protein similar to wheat seed glutenin.

Amino Acid Sequence↗

Effect of ragweed hyperimmune human gamma globulin on in vivo and in vitro parameters of ragweed sensitivity.

The effect of ragweed hyperimmune human gamma globulin upon in vitro and in vivo parameters of ragweed sensitivity was examined. In a double-blind study, 40 ragweed sensitive patients were divided into 2 groups and received either ragweed hyperimmune human gamma globulin (treated) or normal pooled globulin (control). Parameters of ragweed sensitivity studied before and following injections of the gamma globulin included skin test and nasal provocation end point titration, specific IgE antibody as measured by the RAST, leukocyte histamine release, total serum ragweed antibody level, and total serum IgE. Changes in measured parameters varied in both groups of patients. In the "treated" patient group, 1 wk later nasal sensitivity decreased significantly, and there was a trend toward decreased histamine release from leukocytes. No discernable effect was noted upon the other parameters. Thus, with the dosage used, the parenterally administered hyperimmune gamma globulin did not influence most measurements of ragweed hypersensitivity. The concept of this therapeutic approach might warrant further investigation.

Adolescent↗

The elastase inhibitors of swine serum: isolation and partial characterization of the beta 1-globulin inhibitor and its interaction with elastase.

1. The principal elastase inhibitor of swine serum, a beta 1-globulin, has been isolated from serum by ammonium sulfate fractionation, DEAE-cellulose column chromatography and chromatofocusing. 2. The purified beta 1-globulin was homogeneous by immunoelectrophoresis and sodium dodecyl sulfate polyacrylamide gel electrophoresis. Multiple zones (isoinhibitors) were produced on anionic polyacrylamide gels. The mol. wt for the native, elastase-inactivated inhibitor and the beta 1-globulin-elastase complex were respectively, 65,467 and 60,000 and 79,667. The amino acid residue weight was 63,331. 4. The electrophoretic mobilities of the native inhibitor, elastase-inactivated inhibitor and the inhibitor-elastase complex were respectively, -3.4, -3.8 and -2.2 x 10(-5) cm2/V per sec, the isoelectric points were respectively, 4.78-5.28 (major pIs = 5.15, 5.35), 4.63-5.35 (major pI = 5.13) and 6.02-6.2 (major pI = 6.12). 5. The first order dissociation rate constant for the beta 1-globulin-elastase complex (two-fold molar excess of elastase at 37 degrees C) was 1.9 x 10(-3) per sec with complete dissociation in 40.4 min. The dissociation constant for the complex was 1.47 x 10(-7) M. One mol of elastase was bound per mol of the inhibitor. 6. The beta 1-globulin-elastase complex reacts with antibody to either protein moiety.

Animals↗

Multiple binding sites for nuclear factors in the 5'-upstream region of two alpha 2u-globulin genes: implications for hormone-regulated and tissue-specific control.

In order to understand the tissue- and hormone-specific control of alpha 2u-globulin synthesis we isolated the 5'-upstream putative regulatory region of two alpha 2u-globulin genes: RAP 01 and RAO 01. Both clones seem to be expressed in rat liver. DNAseI footprinting analysis after incubation with rat liver nuclear extracts was used to identify regions of potential interest. Particular attention was paid to protected regions located in the neighbourhood of domains which, according to our previous studies, interact specifically with androgen- and glucocorticoid-receptor complexes. Fifteen DNAseI footprints could be mapped in clone RAO 01 (bp -758 up to the cap site). Nineteen footprints were observed in the corresponding region of RAP 01. Differences in the footprinting patterns were mainly observed in the more distal regions. Our data confirm the presence in both clones of two binding sites for the liver enriched factor pseudo-NF1 and one site for C/EBP previously observed in other alpha 2u-globulin genes. In addition we have been able to demonstrate, in RAP 01 only, a binding site for transferrin-liver factor 1. No differences in footprinting patterns could be demonstrated using liver nuclear extracts derived from animals with a high hepatic expression of alpha 2u-globulins (normal male rats) and animals with low to absent expression (prepubertal rats, female rats, rats with the testicular feminization syndrome, diabetic rats and hypophysectomized animals). Transfection experiments indicate that a fragment of RAP 01 (bp -643 up to -617) is able to act as a glucocorticoid and as an androgen response element. Larger fragments of RAP 01 and fragments of RAO 01 are ineffective. It is concluded that the expression of individual alpha 2u-globulin genes is probably the result of combinatorial interactions of several trans-acting factors with appropriate cis-acting elements. Moreover, important sites for tissue-specific and hormone-regulated expression may be situated outside the regions investigated.

Alpha-Globulins↗

Identification and characterization of linear B-cell epitopes of beta-globulin, a major allergen of sesame seeds.

BACKGROUND: The increased consumption of foods containing sesame seeds is paralleled by an increase in reported sesame-induced allergic reactions. OBJECTIVE: This study aimed at identifying and characterizing the linear B-cell epitopes of the 14-kd beta-globulin, the major allergen of sesame seed. METHODS: A peptide containing 71 amino acids (peptide B) was previously identified by us as the IgE-binding region on beta-globulin. To determine the amino acid sequence of the IgE-binding sites on peptide B, we synthesized overlapping peptides 20 and 10 amino acid residues long that span the entire length of peptide B, which were offset from each other by 10 and 2 amino acid residues, respectively. Sera from 20 subjects given diagnoses of allergy to sesame beta-globulin served to identify the epitopes by using the dot-blot test. RESULTS: At least 9 different IgE-recognition sites were identified on peptide B. Three of them, numbers 2, 3, and 13 (corresponding to amino acids 46-55, 48-57, and 76-86, respectively, in the beta-globulin sequence), appeared to be immunodominant IgE-binding epitopes. Also, these peptides were best recognized in terms of intensity of response. There was no obvious sequence motif shared by the 9 different IgE-binding epitopes of beta-globulin. However, approximately 60% of the amino acids represented in the epitopes are hydrophobic residues. CONCLUSION: Identification of the IgE-binding epitopes might provide a better understanding of the functional role the allergens play in the disease and might have implications for immunodiagnosis and probably immunotherapy.

Adolescent↗

Effect of four different oral contraceptives on various sex hormones and serum-binding globulins.

In a double-blind, controlled, randomized, four-arm, bicentric clinical study, the effect of four oral contraceptives (OCs) on various hormone parameters and serum-binding globulins was investigated. Four groups with 25 volunteers each (18-35 years of age) were treated for six cycles with monophasic combinations containing 21 tablets with either 30 microg ethinylestradiol (EE) + 2 mg dienogest (DNG) (30EE/DNG), 20 microg EE + 2 mg DNG (20EE/DNG), 10 microg EE + 2 mg estradiol valerate (EV) + 2 mg DNG (EE/EV/DNG) or 20 microg EE + 100 microg levonorgestrel (LNG) (EE/LNG). The study was completed by 91 subjects. Blood samples were taken after at least 12 h of fasting on Day 21-26 of the preceding control cycle and on Day 18-21 of the first, third and sixth treatment cycle. The serum concentrations of free testosterone were significantly decreased by about 40-60% in all four groups, while those of dehydroepiandrosterone sulfate (DHEAS) showed a time-dependent decrease during treatment. Except for EE/EV/DNG, which increased prolactin significantly during the third and sixth cycles, no change was observed with the EE-containing preparations. There was a significant increase in the levels of serum-binding globulins during treatment, which differed according to the composition of the OCs used. The rise in sex hormone-binding globulin (SHBG) was highest during intake of 30EE/DNG (+320%) and lowest with EE/LNG (+80%), while the effect of 20EE/DNG and EE/EV/DNG was similar (+270%). The thyroxine-binding globulin (TBG) levels increased significantly, by 50-60%, during treatment with the DNG-containing formulations, while the effect of EE/LNG was less significant (+30%). The rise in corticosteroid-binding globulin (CBG), which occurred in all groups, was most pronounced in women treated with 30EE/DNG (+90%) and least with EE/EV/DNG (+55%), indicating a strong influence of EE and no effect of the progestogen component. In all treatment groups, the frequency of intracyclic bleeding rose in the first treatment cycle and decreased thereafter. Cycle control was significantly better with 30EE/DNG or EE/LNG than with 20EE/DNG or EE/EV/DNG. There was no significant change in blood pressure, body mass index or pulse rate throughout the study. In conclusion, the DNG-containing OCs caused a higher rise in SHBG and TBG levels than the LNG-containing preparation. The effects on CBG suggest a lesser hepatic effect of 2 mg EV as compared to 20 or 30 microg EE. In contrast to EE, the use of estradiol in OCs appeared to increase prolactin release, while the cycle control was better with the OC containing 30 microg EE.

Adolescent↗

The effect of monophasic combinations of ethinyl estradiol and norethindrone on gonadotropins, androgens and sex hormone binding globulin: a randomized trial.

The effects of different monophasic combinations of ethinyl estradiol and norethindrone on FSH, LH, sex hormone binding globulin, total testosterone, androstenedione, and dehydroepiandrosterone sulfate levels in non-obese, non-hirsute women were compared. Retrospective analysis of frozen serum from a prospective randomized trial in which women received one of three oral contraceptive pills containing ethinyl estradiol 50 micrograms/norethindrone 1 mg, ethinyl estradiol 35 micrograms/norethindrone 1 mg or ethinyl estradiol 35 g/norethindrone 0.5 mg for nine cycles was conducted. Blood samples were obtained prior to treatment and during the third, sixth and ninth pill cycles. Ethinyl estradiol 50 micrograms/norethindrone 1 mg and ethinyl estradiol 35 micrograms/norethindrone 1 mg suppressed FSH, LH, and total testosterone and increased sex hormone binding globulin to a similar degree. Ethinyl estradiol 35 micrograms/norethindrone 0.5 mg resulted in less suppression of FSH, LH, and total testosterone, but greater elevation of sex hormone binding globulin. Dehydroepiandrosterone sulfate was suppressed to a similar degree with ethinyl estradiol 35 micrograms/norethindrone 1 mg and ethinyl estradiol 35 micrograms/norethindrone 0.5 mg, but ethinyl estradiol 50 micrograms/norethindrone 1 mg resulted in the least suppression of dehydroepiandrosterone sulfate. Ethinyl estradiol 35 micrograms/norethindrone 1 mg caused greater suppression of androstenedione than did the other two oral contraceptives. Oral contraceptive-induced changes in gonadotropins, androgens, and sex hormone binding globulin can be predicted by considering the relative amounts of estrogen and progestin in the pill. When combined with 1 mg of norethindrone, 50 micrograms of ethinyl estradiol did not result in greater suppression of FSH, LH, or total testosterone or in greater elevation of sex hormone binding globulin than did 35 micrograms of ethinyl estradiol.

Adolescent↗