Schistosoma mansoni surface membrane components play a relevant role in the host-parasite interaction, and some are released in vivo as circulating antigens. n-Butanol extraction favors the release of membrane antigens like alkaline phosphatase, which has been shown to be specifically recognized by antibodies from S. mansoni-infected humans and animals. In the present study, components in the n-butanol extract (BE) of the adult S. mansoni worm membrane fraction were separated by one-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis (1D SDS-PAGE [15%]) and further analyzed by immunoblotting (immunoglobulin G) using defined sera. S. mansoni-infected patient sera, but not sera of uninfected patients or sera obtained from patients infected with other parasite species, specifically and variably recognized up to 20 polypeptides in the molecular mass range of ∼8 to >80 kDa. There were some differences in the number, intensity, and frequency of recognition of the BE antigens among sera from Venezuelan sites of endemicity with a different status of schistosomiasis transmission. Antigens in the 28- to 24-kDa molecular mass range appeared as immunodominants and were recognized by S. mansoni-positive sera from all the sites, with recognition frequencies varying between 57.5 and 97.5%. Immunoblotting with BE membrane antigens resulted in a highly sensitive (98.1%), specific (96.1.0%), and confirmatory test for the immunodiagnosis of schistosomiasis in low-transmission areas.
Materials indistinguishable from authentic mono- and diiodotyrosines were identified in extracts of normal human serum as well as in extracts of purified human serum albumin. These materials were not found in association with the other serum proteins. Identification of MIT and DIT was made by a technique using rechromatography to constant specific activity, as well as by the Barker wet ash distillation method, which established the compounds in question as being iodinated ones. By two different extraction and chromatographic methods we estimated the amounts of both MIT and DIT present in normal human serum or albumin; the estimates were in good agreement. These compounds together constituted between 19% and 25% of the extractable serum iodine.
Lithium has been reported to be goitrogenic when used for the treatment of manic-depressive psychosis. To investigate the effects of lithium on iodine metabolism, male Sprague-Dawley rats were placed on a low iodine (LID) or normal iodine diet (NID) containing enough Li2CO3 to give serum lithium levels of 0.23-0.86 mEq/liter (human therapeutic range is 0.6-1.6 mEq/liter). The following effects were noted with lithium treatment: (a) thyroid weight increased concomitant with a slowing of thyroidal iodine release; (b) the ability to concentrate iodide was increased only after goiters were established; (c) on the LID, 131I uptake was elevated throughout all phases of treatment, even when the release rate was normal; (d) iodine organification was unaffected but the proportion of 131I present as iodothyronines was decreased; (e) the thyroidal 127I content was increased; (f) despite these changes, the serum PBI remained normal as did the thyroxine turnover rate; and (g) thyrotropin (TSH) levels in serum were the same as controls except for a slight elevation early in the course of treatment; TSH levels did not correlate with goitrogenesis.
When LiCl was injected in large doses into intact rats (giving serum lithium levels of 3.08-3.89 mEq/liter), the iodide concentrating mechanism, 131I uptake, and 131I release rates were depressed. Similar experiments in hypophysectomized rats receiving TSH demonstrated these to be local antithyroid effects not mediated through the pituitary.
The discrepancy between acute and chronic responses to lithium, and the dissociation between the inhibition of iodine release and stimulatory effects is discussed.
The problem as to whether iodohistidines are normally biosynthetized in thyroglobulin and thyralbumin has been examined both in man and the rat. Evidence has been obtained for the first time that diiodohistidine (DIH) is present in both species in these two iodoproteins. The biosynthesis of monoiodohistidine (MIH) in the thyroglobulin of the normal rat has been confirmed and extended to rat thyralbumin and to human thyroid iodoproteins.
The iodohistidine identification is based on five original methods including: (a) the preparation of stable and radioiodine-labeled iodohistidines; (b) the protection of the labile iodohistidines during the iodoprotein enzymatic hydrolysis; (c) the isolation of iodohistidines by ion-exchange resin chromatography; (d) their separation from each other and from iodinated cationic butanol-insoluble compounds by Sephadex G-10 chromatography; and (e) their purification by successive crystallizations to a constant specific activity.
Iodohistidine levels (in percent of protein radioactivity from iodide given in vivo) were found comparable in man and the rat. However, the values (mean ±SE) for thyroglobulin (MIH, 0.61±0.10%; DIH, 0.050±0.015%) and for thyralbumin (MIH, 2.61±0.57%; DIH, 0.28±0.09%) differ significantly (P < 0.05).
Iodohistidines are stable during in vitro exposure to iodotyrosine dehalogenase preparations. In contrast to iodotyrosines the iodohistidines when given in vivo to man either orally or intravenously were in large part recovered in 24-h urines.
Alkaline phosphatase has been extracted from human pancreas with n-butanol and has been shown to have distinctive isoenzyme characteristics when compared with the enzymes of small intestine and of normal serum. The enzyme has been demonstrated histochemically in a carcinoma of the pancreas and also in an islet cell tumour and the tumour enzyme has been shown to be unique, not only electrophoretically, but in being remarkably heat sensitive and in being activated by low molarities of urea.
Lactic acid bacteria (LAB) are beneficial probiotic organisms that contribute to improved nutrition, microbial balance, and immuno-enhancement of the intestinal tract, as well as anti-tumor activity. The aim of the present work was to study the growth inhibition of tumor cells by butanol extract of Bifidobacterium adolescentis isolated from healthy young Koreans.
The anti-proliferative activity of B. adolescentis isolates was assessed by XTT assays on three human colon cancer cell lines (Caco-2, HT-29, and SW480). The effects of B. adolescentis SPM0212 butanol extract on tumor necrosis factor-α (TNF-α) and nitric oxide (NO) production were tested using the murine macrophage RAW 264.7 cell line.
The butanol extract of B. adolescentis SPM0212 dose-dependently inhibited the growth of Caco-2, HT-29, and SW480 cells by 70%, 30%, and 40%, respectively, at 200 μg/mL. Additionally, the butanol extract of B. adolescentis SPM0212 induced macrophage activation and significantly increased the production of TNF-α and NO, which regulate immune modulation and are cytotoxic to tumor cells.
The butanol extract of B. adolescentis SPM0212 increased activity of the host immune system and may improve human health by helping to prevent colon cancer as a biological response modifier.
We have found whole human platelets, granulocytes, and mononuclear leukocytes to possess high affinity for the toxic lipopolysaccharide from all gram-negative bacteria tested. We have extracted these cells and platelets with n-butanol-water; all endotoxin-binding activity resided in the organic phase. These endotoxin-binding extracts did not block serologically active groupings on endotoxins or receptors on the erythrocytes. The specificity of these still crude materials was less that that of the highly purified erythrocyte lipopolysaccharide receptor previously described by us, since they bound some bacterial antigens not related to endotoxins. Depending on source, the n-butanol extracts contained 40 to 52% glycerophosphatides (most active), 15 to 22% sphingomyelin, 17% cholesterol, less than 2 to 5% triglycerides, and 7 to 13% inactive peptide. The most active substances in the n-butanol extract were soluble in petroleum ether, whereas the peptide and sphingomyelin were not. Thus, no constituent protein, carbohydrate, or nucleic acid was present in the most highly active material. Polyacrylamide gel electrophoresis of the petroleum ether-soluble material showed for each extract one lipid band only, which was well defined and migrated similarly to phosphatidyllipids. Because of the lipidic nature of the inhibitory substances from leukocytes and platelets we also tested the lipid A component of bacterial endotoxins and some of its derivatives. Lipid A inhibited endotoxin coating of erythrocytes. De-O-acylation of lipid A left amide-linked 3-D-hydroxymyristic acid intact and increased the inhibitory activity of lipid A 20-fold. Complete de-O- and de-N-acylation destroyed its inhibitory effect.
Therapies for peanut allergy (PNA) are urgently needed. Food Allergy Herbal Formula -2 (FAHF-2) has profound therapeutic effects in a murine peanut allergy model and is safe for food allergic adults in clinical trials. However the large FAHF-2 pill-load is not conducive to clinical studies in children. Thus refining FAHF-2 to decrease pill-load is essential for the inclusion of children in clinical trials and to facilitate studying FAHF-2 as a clinically useful botanical drug.
Testing long term efficacy and safety of a butanol-purified extract of FAHF-2 (B-FAHF-2) in a murine model of PNA, and to explore its immunological mechanisms of action.
FAHF-2 was purified by butanol extraction. C3H/HeJ mice with established PNA received the 1st course of B-FAHF-2 at 6 mg, twice daily for 7 weeks (PNA/B-FAHF-2) or water (PNA/Sham) and were then challenged immediately after completing the treatment and 6 more times every 1–2 months post treatment up to week 50. Mice then received a second course of B-FAHF-2 treatment at week 52 and were challenged at week 65. In vivo and in vitro immunological effects on T, B and mast cells were also determined.
Butanol purification reduced the volume of the effective dose ~5 fold. All PNA/B-FAHF-2 mice were completely protected from peanut anaphylaxis until the 5th challenge after the 1st course of treatment, as compared to PNA/sham mice. Partial protection persisted up to 50 weeks. A 2nd treatment course restored complete protection. B-FAHF-2 significantly suppressed Th2 cytokine, IgE and histamine levels in vivo, and showed direct inhibition of Th2, IgE-producing B cells and mast cell activation in vitro. B-FAHF-2 had a high margin of safety.
Conclusion and clinical relevance
B-FAHF-2 produced long-lasting protection against PN anaphylaxis for approximately half of the murine lifespan without side effects. B-FAHF-2 exhibited direct effects on multiple food allergy effector cells.
Chinese herbal medicine formula; FAHF-2; B-FAHF-2; Peanut Anaphylaxis; Th2 cytokines; Histamine; IgE
The aim of this research was to determine the intensity and mechanisms of the cytotoxic actions of five extracts isolated from the endemic plant species Helichrysum zivojinii Černjavski & Soška (family Asteraceae) against specific cancer cell lines. In order to evaluate the sensitivity of normal immunocompetent cells implicated in the antitumor immune response, the cytotoxicity of extracts was also tested against healthy peripheral blood mononuclear cells (PBMC).
The aerial parts of the plants were air-dried, powdered, and successively extracted with solvents of increasing polarity to obtain hexane, dichloromethane, ethyl-acetate, n-butanol and methanol extracts. The cytotoxic activities of the extracts against human cervix adenocarcinoma HeLa, human melanoma Fem-x, human myelogenous leukemia K562, human breast adenocarcinoma MDA-MB-361 cells and PBMC were evaluated by the MTT test. The mode of HeLa cell death was investigated by morphological analysis. Changes in the cell cycle of HeLa cells treated with the extracts were analyzed by flow cytometry. The apoptotic mechanisms induced by the tested extracts were determined using specific caspase inhibitors.
The investigated Helichrysum zivojinii extracts exerted selective dose-dependent cytotoxic actions against selected cancer cell lines and healthy immunocompetent PBMC stimulated to proliferate, while the cytotoxic actions exerted on unstimulated PBMC were less pronounced. The tested extracts exhibited considerably stronger cytotoxic activities towards HeLa, Fem-x and K562 cells in comparison to resting and stimulated PBMC. It is worth noting that the cytotoxicity of the extracts was weaker against unstimulated PBMC in comparison to stimulated PBMC. Furthermore, each of the five extracts induced apoptosis in HeLa cells, through the activation of both intrinsic and extrinsic signaling pathways.
Extracts obtained from the endemic plant Helichrysum zivojinii may represent an important source of novel potential antitumor agents due to their pronounced and selective cytotoxic actions towards malignant cells.
Helichrysum zivojinii; Cytotoxicity; Cancer cells; Peripheral blood mononuclear cells; Apoptosis
We studied two sisters who developed large non-toxic goitres in adolescence. Deiodinase deficiency was diagnosed by a rapid thyroid uptake of radioactive iodine (RAI) at 2 hours associated with a marked fall in thyroidal 131I by 24 hours. Serial RAI scans in the second patient documented evolution of the iodine-deficient state. Conservation of intra-thyroidal iodine stores was maintained by avid iodine uptake and failure to release organified 131I. With progressive loss of inorganic iodine, hypothyroidism developed, associated with a rise in serum TSH which further exacerbated the loss of iodine. Treatment with L-thyroxine resulted in an improvement of thyroid function, but normalization was achieved only after small doses of Lugol's iodine were administered. These studies illustrate the variable nature and late onset of an inborn error of thyroid metabolism. This family supports an autosomal recessive mode of inheritance for deiodinase deficiency. We have documented progression from a euthyroid to hypothyroid state resulting from decompensation of iodine conservation mechanisms.
Ageratum conyzoides has been used in folklore for the treatment of a wide range of diseases. In the present investigation, the in vitro activity of ethanol, petroleum ether, ethylacetate, butanol, and water extracts of A. conyzoides were screened in some cancer cell lines using the sulphorhodamine B (SRB) assay. These cell lines include: Human non-small cell lung carcinoma (A-549), human colon adenocarcinoma (HT-29), human gastric carcinoma (SGC-7901), human golima (U-251), human breast carcinoma (MDA-MB-231), human prostate carcinoma (DU-145), human hepatic carcinoma (BEL-7402), and mouse leukemia (P-388) cancer cell lines. Furthermore, kaempferol was isolated from the ethylacetate extract and the structure was elucidated by nuclear magnetic resonance (NMR) and mass spectroscopy. The effect of DPPH antiradical activity on the extracts and kaempferol was also determined. The results showed that ethylacetate extract exhibited the highest cytotoxic activity on A-549 and P-388 cancer cells with IC50 values of 0.68 and 0.0003 μg/ml, respectively. Kaempferol isolated from the ethylacetate extract of A. conyzoides rapidly scavenged DPPH at a concentration of 130.07 ± 17.36 g/kg. The result therefore showed that A. conyzoides possessed anticancer and antiradical properties.
Ageratum conyzoides; anticancer; antiradical scavenging activity; cytotoxicity
The Ukinga and Uwanji regions, located in the southern highlands of Tanzania, were studied for the degree of iodine deficiency and the incidence of goitre and hypothyroidism, respectively. A urinary iodine excretion as low as 17.6 +/- 9.3 micrograms/g creatinine was observed in Wangama village. The mean goitre prevalence in 27 villages in Uwanji ranged between 65 and 96% (n = 3031 schoolchildren). Of 681 pregnant women from Ukinga 79.6% had goitre. The prevalence of cretinism as estimated on clinical criteria was 3% in Magoye (Uwanji). A normal serum TSH (below 2.1 mU/l) was observed in only 12 out of 66 school children before iodine prophylaxis, whereas the T4/TBG ratio was decreased in 36 of 63 cases. Blood spot TSH levels in newborn infants (n = 219) from mothers without iodine supplementation were above 12 mU/l in 45%. In contrast, only 20.3% of the newborn (n = 118) had elevated blood spot TSH (p less than 0.002) when the mothers had received an iodised oil injection during pregnancy. Most of the newborn (n = 18; 75%) of the latter group with elevated TSH (n = 24) came from mothers who had received the iodine injection only 1-25 days before delivery. Maternal iodine prophylaxis in late pregnancy does not increase the rate of neonatal hypothyroidism. Conclusions: It has been confirmed that severe iodine deficiency resulting in endemic goitre, cretinism, and hypothyroidism is prevalent in the regions studied. Dried blood spot TSH determinations may serve as an index for the efficiency of iodine prophylaxis programmes. Such a programme was carried out with relatively little expenditure and effort on a large scale basis.
A series of 105 patients treated at least two years earlier with radioactive iodine for thyrotoxicosis have been surveyed. Eighty-five patients (81%) were euthyroid clinically and on the basis of routine thyroid function tests. Of the euthyroid patients 46 (54%) had normal thyroid-stimulating hormone (TSH) levels and 39 (46%) had raised TSH levels. There was no difference in serum triiodothyronine levels between these two groups but the serum protein bound iodine and serum thyroxine, though still well within the normal range, were significantly lower in the group with raised TSH levels. The serum cholesterol was also significantly higher in this latter group.
Most of the euthyroid patients were seen again a year later. None had become hypothyroid and neither those with normal nor those with raised TSH levels showed any evidence of a decline in the level of serum thyroxine.
It is concluded that raised serum TSH levels in patients treated with iodine-131 are not necessarily indicative of hypothyroidism. There is no indication that patients who have this abnormality become overtly hypothyroid over a 12-month follow up.
Iodine is essential for normal fetal and neonatal development. We studied the prevalence and impact on fetal thyroid development of iodine deficiency in pregnant women in the northern part of the Paris conurbation.
Materials and Methods
110 patients underwent several determinations of urinary iodine excretion (UIE) and of serum FT4, FT3, and TSH. Fetal thyroid gland size was assessed using ultrasonography.
We found evidence of widespread iodine deficiency (mean UIE, 49.8 µg/L [standard deviation, 2.11]). Iodine deficiency did not correlate significantly with maternal thyroid parameters but showed a significant negative correlation with fetal thyroid gland size (rho = 0.25, P = 0.02).
Iodine deficiency during pregnancy is still a problem in our geographical area and affects the fetal thyroid gland.
Clinical Trials.gov NCT00162539
The efficacy of decoction in extracting mycobactericidal compounds from Flourensia cernua (Hojasé) leaves and fractionation with solvents having ascending polarity was compared with that of (i) ethanol extraction by still maceration, extraction with a Soxhlet device, shake-assisted maceration, or ultrasound-assisted maceration, followed by fractionation with n-hexane, ethyl acetate, and n-butanol; (ii) sequential extraction with n-hexane, ethyl acetate, and n-butanol, by still maceration, using a Soxhlet device, shake-assisted maceration, or ultrasound-assisted maceration. The in vitro mycobactericidal activity of each preparation was measured against drug-sensitive (SMtb) and drug-resistant (RMtb) Mycobacterium tuberculosis strains. The results of which were expressed as absolute mycobactericidal activity (AMA). These data were normalized to the ΣAMA of the decoction fraction set. Although decoction was inactive, the anti-RMtb normalized ΣAMA (NAMA) of its fractions was comparable with the anti-RMtb NAMA of the still maceration extracts and significantly higher than the anti-SMtb and anti-RMtb NAMAs of every other ethanol extract and serial extract and fraction. Hexane extracted, from decoction, material having 55.17% and 92.62% of antituberculosis activity against SMtb and RMtb, respectively. Although the mycobactericidal activity of decoction is undetectable; its efficacy in extracting F. cernua active metabolites against M. tuberculosis is substantially greater than almost all pharmacognostic methods.
A method is described for the determination of serum thyroxine using ion-exchange resin sponges. The principle is based on the use of protein-binding sites and part of the technique is similar to the Triomet resin uptake test. An isotope extraction recovery procedure is incorporated in the method. Studies of factors influencing the analysis are reported and the results of tests of accuracy, precision, and specificity are presented. Serum thyroxine iodine values are compared with protein-bound iodine estimations. The values found in health and disease are similar to the results of previous workers.
The specificity of the analysis represents a considerable advantage over protein-bound iodine determinations since no interference is caused by iodides, iodine-containing contrast media, mercurial diuretics, mono-iodotyrosine, di-iodotyrosine, di-iodothyronine, and also tri-iodothyronine in physiological amounts. Therapy with diphenylhydantoin and tri-iodothyronine will tend to depress protein-bound iodine levels and thyroxine values are similarly influenced.
Trichloroacetic acid extracts of plasma were fractionated on a CG-50 resin column and the 50% acetic acid eluents chromatographed on silicic acid-impregnated glass paper in butanol-acetic acid-water. The specific arginine vasopressin (AVP) zone was eluted and assayed for antidiuretic activity in the diuretic rat. Thioglycolate inactivation was used to confirm AVP activity. Recovery of as little as 4 μU AVP per ml plasma ranged between 80 and 90%. In normal subjects after an overnight fast, plasma AVP ranged between 2.5 and 10.0 μU per ml. AVP secretion was inhibited by hemodilution and stimulated with nicotine and hypertonic saline. Plasma AVP was absent in patients with diabetes insipidus even after neurohypophyseal stimulation. Plasma AVP was abnormally elevated during mild dehydration and remained above the normal range despite hemodilution in patients with untreated adrenocortical insufficiency demonstrating a delayed water diuresis. Glucosteroid therapy lowered plasma AVP to normal in dehydrated patients. A normal diuretic response to hydration was accompanied by a fall in plasma AVP to zero in steroid-treated patients. These findings suggest that hypersecretion of AVP may play an important role in the abnormal water metabolism of adrenocortical insufficiency and that the glucosteroids promote normal water diuresis by inhibiting the secretion of AVP from the neurohypophysis.
Pregnancy has a variety of effects on maternal thyroid function. Thyroid gland enlargement is common particularly in areas of relative iodine deficiency. The renal clearance of iodine is increased in pregnancy and together with an increased volume of iodine distribution, leads to a low plasma inorganic iodine and thus increases the thyroidal iodine clearance. However, the absolute iodine uptake and hormone production rate remain unchanged. There is an increase in the serum thyroxine (T4)and triiodothyronine (T3) concentration largely due to an increase in thyroid hormone-binding proteins. Free thyroxine and free T3 remain unchanged in pregnancy as does the Free Thyroxine Index, which gives the single most accurate measure of thyroid function. The placenta secretes a number of thyroid stimulators including human chorionic gonadotrophin and possibly chorionic thyrotrophin and molar thyrotrophin whose physiological role is to date poorly understood. The fetal thyroid develops independently, and although fetal T4 CONCENTRATION RISES PROGRESSIVELY To maternal by term, the T3 concentration is markedly reduced owing to preferential formation of inactive reverse T3.
The development of theranostic agents with high detection sensitivity and antitumor efficacy at low concentration is a challenging task for target selective imaging and therapy of cancers. In this study, folate-conjugated and radioactive-iodine-labeled gold nanorods (GNRs) were designed and synthesized for target selective SPECT/CT imaging and subsequent thermal ablation of folate-receptor-overexpressing cancers. Both (ortho-pyridyl) disulfide-poly(ethylene glycol)-folate and a short peptide, H2N-Tyr-Asn-Asn-Leu-Ala-Cys-OH, were conjugated on the surface of the GNRs through thiol chemistry. The tyrosine in the peptide sequence was introduced for radioactive-iodine labeling through an iodine-tyrosine interaction. The labeling efficiency of radioactive iodine was more than 99%. Radiochemical stability tests on iodine-125-labeled GNRs in human serum showed that 91% of the iodine-125 remained intact on the GNRs after incubation for 24 h. In vitro and in vivo results in this study confirmed the potential utility of folate-conjugated and iodine-125-labeled GNRs as smart theranostic agents. This type of platform may also be useful for the targeted SPECT/CT imaging and photothermal therapy of inflammatory diseases such as atherosclerosis and arthritis, in which folate-receptor-overexpressing macrophages play pivotal roles.
Target selective imaging; gold nanorods; SPECT/CT; photothermal therapy
Tobacco smoking is associated with an increased risk of cancer in a number of organs, including bladder and lung. Tobacco smoke contains at least 50 known chemical carcinogens that exert their biological effects through their covalent binding to cellular DNA. Examining human DNA for the presence of altered nucleotides is a means of monitoring exposure to genotoxic chemicals. DNA isolated from 73 human bladder biopsies has been analyzed by 32P-postlabeling for the presence of aromatic/hydrophobic adducts. Butanol extraction of DNA digests resulted in up to a 3-fold greater recovery of adducts than nuclease P1 digestion. Among 16 nonsmokers, adduct levels were in the range 3.2-20.8/10(8) nucleotides (mean 9.7). Eight ex-smokers had values in the range 2.6-12.3 (mean 7.1). Thirteen smokers had adduct levels between 1.3 and 26.7 adducts/10(8) nucleotides (mean 9.5, not different from nonsmokers). Six cigar smokers had higher levels of adducts (mean 12.1, range 7.3-15.0), but pipe smokers did not (five samples, mean 8.6, range 2.9-12.7). A further 8 samples from nonsmokers and 17 from smokers were examined in more detail. Although most of the DNA binding appears not to be smoking related, the levels of one adduct were found to be on average 2-fold higher in smokers (p < 0.005, one-tailed t test). Studies on tissues of the respiratory tract demonstrate a correlation between DNA adduct levels and exposure to tobacco smoke. Evidence to date on the influence of smoking on adducts in peripheral blood cells is equivocal; some studies demonstrate a significant effect, whereas others do not.(ABSTRACT TRUNCATED AT 250 WORDS)
An important polysaccharide, amylose crystallizes as a regular single left-handed helix from a propanol, butanol or iodine solution. However, its solution structure remains elusive because amylose does not form molecular solutions in these solvents, and standard spectroscopic techniques cannot be exploited to determine its structure. Using the AFM, we forced individual amylose chains adsorbed to a surface to enter these poor solvents and carried out stretch-release measurements on them in solution. In this way, we directly captured the formation of individual amylose helices induced by butanol and iodine. With an accuracy approaching that of X-ray diffraction on amylose crystals, we determined that the pitch of the helix in solution is 1.3 Å/ring. We also directly measured the force driving the formation of the helix in solution to be 50 pN. SMD simulations in explicit butanol reproduced the AFM-measured force-extension curves and revealed that the long plateau feature is caused by the rupture of O(2)n-O(6)n+6 and O(3)n-O(6)n+6 hydrogen bonds and by the unwinding of the helix. We also found that amylose helices formed in iodine solution are more compliant and hysteretic as compared to helices in butanol, which extend/relax reversibly. In iodine solution, the formation of the helix is inhibited by force and limited by the slow kinetics of the amylose-iodine complex. By forcing individual molecules into poor solvents and performing force spectroscopy measurements in solution, our AFM approach uniquely supplements X-ray diffraction and NMR methods for investigating solution conformations of insoluble biopolymers.
Butanol-insoluble iodinated compounds in the urine of patients with congenital goiters have been generally regarded as iodopeptides. Monoiodohistidine (MIH) and diiodohistidine (DIH) were identified from the urine of four patients with congenital goitrous hypothyroidism. From radioiodine studies, 40-70% of the urinary radioactivity was in the iodide-free fraction from which about 40% was identified as MIH and DIH by crystallizations to a constant specific activity.
Iodotyrosines were simultaneously identified in the urine. However the presence of an iodotyrosine-deiodinase activity was demonstrated in the two removed goiters with a normal Km for MIT. In vivo iodotyrosine deiodination was normal for hypothyroid subjects.
No thyroglobulin was identified in the thyroids from these patients. The major iodoprotein was iodoalbumin which, after in vivo labeling, contained 84-89% of the total soluble protein radioactivity. The thyroxine content of the goiter iodoalbumins and other iodoproteins was extremely low.
Iodohistidines were identified in comparable proportions in the iodoalbumin and in the other iodoproteins isolated from each goiter. The average iodohistidine content of these proteins as crystallizable MIH and DIH was in the individual cases 15 and 4% of the in vivo incorporated radioiodine. DIH was identified in all iodoprotein fractions. The mean DIH/MIH ratios from the individual cases were 1.16 and 0.35. The corresponding DIT/MIT ratios were 3.19 and 1.45, respectively.
The major consequence of this thyroglobulin defect is the iodination of inappropriate proteins (mainly albumin) resulting in low yields of thyroxine and high yields of iodohistidines. Iodohistidines from the goiter iodoproteins were not deiodinated and, at least for MIH, were quantitatively excreted in the urine of these patients. From the MIH iodoalbumin content and the MIH urinary excretion, goiter iodoalbumin turnover estimates were made and, although elevated, could not maintain a normal thyroxine secretion.
The urinary excretion of iodohistidines easily demonstrated by column chromatography is offered as a test for detecting this variety of congenital goiter.
A protein with the electrophoretic, immunologic, and hormone-binding properties of thyroxine-binding globulin (TBG) has been prepared from human plasma and labeled with radioiodine (125-I) by an enzymatic method of iodination. The [125-I]TBG retained the electrophoretic and immunologic characteristics of unlabeled TBG but exhibited a partial loss of thyroxine-binding activity, as assessed by affinity chromatography. The in vivo behavior of [125I]TBG was studied in six euthyroid subjects (controls) with normal serum levels of TBG as measured both by radioimmunoassay and by determination of maximal T4-binding capacity and in four male patients with untreated primary hyperthyroidism, three of whom had elevated serum TBG. The half-time of the final slope of the plasma disappearance curve averaged 5.0 days plus or minus 1.2 (SD) in the controls and ranged from 3.9 to 109 days in the hypothyroid patients. The distribution volume was similar in the two groups, 6.7 plus or minus 1.3 vs. 7.1 plus or minus 2.1 liters. The catabolic clearance rate averaged 0.99 plus or minus 0.33 liters plasma/24 h in the controls and 0.92 plus or minus 0.46 in the hypothyroids. The absolute turnover rate of TBG, calculated from the catabolic clearance rate multiplied by the serum concentration of radioimmunoassayable TBG, averaged 17.8 plus or minus 2.1 mg/day in the controls and ranged from 14.8 to 33.2 mg/day in the hypothyroids. Among the entire group of subjects there was no correlation between the serum TBG concentration and the absolute turnover rate of TBG.
Hypothyroidism from iodide transport deficiency is a rare disease, especially when found in two affected siblings. Treatment with high doses of iodide has been recommended, but no long term results have been reported. Two siblings with congenital hypothyroidism due to total failure to transport iodide have been followed up during twelve and a half years of treatment with oral potassium iodide. Iodine doses varied between 10.3 and 22 mg/day, and serum total iodine concentrations between 100 and 210 micrograms/dl. Total triiodothyronine (T3), thyroxine (T4) and free T4 were in the normal range during the time of study. Basal thyroid stimulating hormones (TSH) and maximum TSH response to thyrotrophin releasing hormone (TRH) were also in the range of normal values. These data along with clinical findings confirmed the potential usefulness of iodine in hypothyroidism due to complete iodide transport defect.
Extractive fermentation has been proposed to enhance the productivity of fermentations that are end product inhibited. Unfortunately, good extractants for butanol, such as decanol, are toxic to Clostridium acetobutylicum. The use of mixed extractants, namely, mixtures of toxic and nontoxic coextractants, was proposed to circumvent this toxicity. Decanol appeared to inhibit butanol formation by C. acetobutylicum when present in a mixed extractant that also contained oleyl alcohol. However, maintenance of the pH at 4.5 alleviated the inhibition of butanol production and the consumption of butyrate during solventogenesis. A mixed extractant that contained 20% decanol in oleyl alcohol enhanced butanol formation by 72% under pH-controlled conditions. The production of acetone and acetoin was also increased, even though these two products were not extractable. The enhancement of butanol formation was not limited by the toxicity of decanol. Supplementation of glucose and butyrate in the extractive fermentation yielded a 47% increase in butanol. The enhancement of butanol formation appeared to be dependent on the presence of dissolved decanol in the broth but was not observed unless an organic phase was present to extract butanol. A mechanism for the effects of decanol on product formation is proposed.