In vitro evidence that carbohydrate moieties derived from uromodulin, an 85,000 dalton immunosuppressive glycoprotein isolated from human pregnancy urine, are immunosuppressive in the absence of intact protein

Our laboratory recently reported the purification of a unique immunosuppressive glycoprotein isolated from human pregnancy urine (7). This glycoprotein, which we term uromodulin, has a m.w. of 85,000 as assessed on SDS-PAGE and is 30% carbohydrate. Uromodulin blocks in vitro antigen-specific T cell proliferation to recall antigens such as tetanus toxoid at concentrations as low as 100 pM. This glycoprotein also blocks the in vitro generation of spontaneous monocyte-mediated cytotoxicity (7, 36). Recent evidence strongly suggests that the primary action of uromodulin is to act as a specific ligand and modulator of IL 1 (10, 33). We now report additional biochemical characterization of uromodulin, and based on three independent lines of evidence, find that its immunologic activity appears to result from its glycosylation. First, measures to alter the tertiary folding of the protein backbone of uromodulin, including succinylation or reduction and carboxymethylation, fail to significantly affect its in vitro bioactivity. Second, after extensive digestion of intact uromodulin with pronase, the majority of the in vitro bioactivity can be recovered in a single carbohydrate-rich fraction. Finally, digestion with N-glycanase (N-glycosidase F-, an enzyme specific for N-asparagine-linked oligosaccharides) and subsequent purification on thin layer chromatography yields a single complex oligosaccharide that appears to be responsible for the majority of the in vitro immunosuppression mediated by uromodulin. These data suggest that uromodulin displays N-linked carbohydrate sequences capable of down-regulating antigen-specific T cell responses in vitro. It has been suggested that endogenous lectins may play an important role as recognition molecules in mammalian, as well as more primitive immune systems (23, 24). Our in vitro biologic data strongly suggest that the carbohydrate portion of uromodulin is an excellent candidate to function as a potential lectin receptor.     

Thyroglobulin interactions with thyroid membranes. Relationship between receptor recognition of N-acetylglucosamine residues and the iodine content of thyroglobulin preparations

Bovine thyroglobulin has been subjected to sequential glycohydrolase treatment in order to define further the components of the carbohydrate chain which are important in binding of the glycoprotein to bovine thyroid membranes. Preparations of asialoagalactothyroglobulin exhibit the best binding, suggesting that exposed N-acetylglucosamine residues on the B carbohydrate chain of thyroglobulin play an important role in the interaction of thyroglobulin with the thyroid membranes. Enhanced binding of asialoagalactothyroglobulin to microsomal, lysosomal, and Golgi membranes, as well as to thyroid cells in culture, was also observed. Isopycnic rubidium chloride gradient centrifugation, a procedure used in the isolation of thyroglobulin molecules with a low iodine content, also isolates thyroglobulin molecules with a low sialic acid content and with an increased ability to interact with wheat germ agglutinin, a lectin which recognizes exposed N-acetylglucosamine residues. The studies further indicate that there is a correlation between iodine content, exposed N-acetylglucosamine residues, and the binding of thyroglobulin to thyroid membranes.     

PHYTOPLANKTON LIPIDS: INTERSPECIFIC DIFFERENCES AND EFFECTS OF NITRATE,SILICATE AND LIGHT-DARK CYCLES1

The lipid content of various phytoplankton species was measured in response to nitrogen and silicon limitation and over the cell cycle in synchronized cultures. In a survey of 30 species it was found that during log-phase growth, green algae contained an average of 17.1% total lipids (% of total dry weight), whereas diatoms contained an average of 24.5%. Nitrogen deprivation for 4 to 9 days resulted in 2- to 3-fold increases in the lipid content of green algae, whereas both increases and decreases were noted in diatoms, depending on the species. The greatest lipid content measured in the study was 72% in Monallantus salina (strain GSB Sticho) which had been deprived of nitrogen for 9 days. Nitrate replenishment in a nitrogen starved culture of Oocystis polymorpha Groover & Bold showed that the excess cellular lipids do not rapidly disappear during recovery, until cell division occurs. A silicate deprivation experiment with Cyclotella cryptica Reimann, Lewin & Guillard (strain 7c) showed an increase in the total cellular lipid fraction from. 30 to 42% of dry weight within 6 h of the onset of silicon limitation, while the mass of lipid material per cell doubled within 12 h. The total lipid fraction in O. polymorpha was found to remain constant over the cell cycle in synchronized cultures regardless of the light regime. The data presented provided the first internally consistent study of phytoplankton lipids for a wide range of species and several growth conditions.     

Regulation of Muscarinic Acetylcholine Receptor Concentration in Cloned Neuroblastoma Cells

Abstract: The concentration of muscarinic acetylcholine receptors in the neuroblastoma cell line NIE-115 is regulated by receptor activation. Muscarinic agonists cause a time and dose-dependent loss of [³H]quinuclidinyl benzilate binding sites from cultured cells. Muscarinic antagonists have no effect on receptor concentration and block agonist-induced regulation. The maximum decrease in steady state receptor levels is 80% and occurs within 9 h. The altered steady state concentration persists as long as agonist remains present. Upon withdrawal of agonist, the concentration of receptors returns to control levels. This increase requires protein synthesis. Kinetically, the increase in receptors following withdrawal of agonist is slower than the decrease caused by addition of agonist, suggesting that bursts of receptor activation could lower receptor levels. In harmony with this prediction, cycles in which receptors are active for 15 min and then inactive for 15 min cause a 50% decrease in receptor concentration in a 6-h period.     

Binding of thyroglobulin to bovine thyroid membranes. Role of specific amino acids in receptor recognition

Bovine thyroglobulin was treated with increasing ratios of succinic anhydride, trinitrobenzene sulfonic acid, tetranitromethane, and N-acetylimidazole in an attempt to assess the role of lysine or tyrosine residues in binding to thyroid membrane receptors. Extensive succinylation results in dissociation to 12 S thyroglobulin with retention of a considerable portion of the three-dimensional structure. Only 25% of the lysine residues can be modified by trinitrophenylation without affecting inter-subunit interactions. Succinylation as well as trinitrophenylation increases the affinity of thyroglobulin for the membrane receptor by a factor of 2. The binding of thyroglobulin to the membrane was reduced after nitration of 30% of the tyrosyl residues with tetranitromethane. O-Acetylation of 40-70% of the tyrosyl residues by N-acetylimidazole nearly abolished the ability of thyroglobulin to bind to the membrane. Removal of the O-acetyl group with hydroxylamine restored the binding properties. The results indicate that tyrosyl residues play an important role in thyroglobulin interactions with thyroid membranes.     

Rapid intraoperative insulin assay: a novel method to differentiate insulinoma from nesidioblastosis in the pediatric patient

Background

The essential objectives for thyroidectomy are: avoidance of injury to the recurrent laryngeal nerves, conservation of the parathyroid glands, an accurate haemostasis and an excellent cosmesis. In the last 10 years major improvements and new technologies have been proposed and applied in thyroid surgery; among these mini-invasive thyroidectomy, regional anaesthesia and intraoperative neuromonitoring, and new devices for achieving dissection and haemostasis. Minor bleeding from small vessels could be a major complication in thyroid surgery. The purpose of ligating vessels is to maintain the surgical site free from an excess of blood and reduce blood loss in the patient.

Materials and methods

Hydroxylated polyvinyl acetal tampons (HPA) are made by a synthetic, open cell foam structure able to absorb fluids up to 25 times the initial weight. We tested their efficacy for small bleeding control and tissue dissection during several thyroid procedures.

Results

HPA tampons have been found extremely useful to absorb blood coming from minor and diffuse loss, helping to control bleeding by a combined action of fluid absorption and local compression. The porous design of the tampon allows the use of the suction device right through the tampon itself. Thanks to the initial mildly hard consistency, we also used HPA tampons as dissecting instruments.

Conclusion

In our experience the use of HPA tampons resulted extremely efficient for minor bleeding control, fluids removal and tissue dissection during thyroid surgery.     

The Role of FilGAP-Filamin A Interactions in Mechanoprotection

Cells in mechanically active environments are subjected to high-amplitude exogenous forces that can lead to cell death. Filamin A (FLNa) may protect cells from mechanically induced death by mechanisms that are not yet defined. We found that mechanical forces applied through integrins enhanced Rac-mediated lamellae formation in FLNa-null but not FLNa-expressing cells. Suppression of force-induced lamella formation was mediated by repeat 23 of FLNa, which also binds FilGAP, a recently discovered Rac GTPase-activating protein (GAP). We found that FilGAP is targeted to sites of force transfer by FLNa. This force-induced redistribution of FilGAP was essential for the suppression of Rac activity and lamellae formation in cells treated with tensile forces. Depletion of FilGAP by small interfering RNA, inhibition of FilGAP activity by dominant-negative mutation or deletion of its FLNa-binding domain, all resulted in a dramatic force-induced increase of the percentage of annexin-V–positive cells. FilGAP therefore plays a role in protecting cells against force-induced apoptosis, and this function is mediated by FLNa.     

Characterization of phosphate residues on thyroglobulin

Follicular 19 S thyroglobulin (molecular weight 660,000) from rat, human, and bovine thyroid tissues contains approximately 10-12 mol of phosphate/mol of protein. These phosphate residues can be radiolabeled when rat thyroid hemilobes, FRTL-5 rat thyroid cells, or bovine thyroid slices are incubated in vitro with [32P]phosphate. Thus labeled, the [32P]phosphate residues comigrate with unlabeled 19 S follicular thyroglobulin on sucrose gradients and gel filtration columns; are specifically immunoprecipitated by an antibody preparation to rat or bovine thyroglobulin as appropriate; and co-migrate with authentic 19 S thyroglobulin when subjected to analytic or preparative gel electrophoresis. Tunicamycin prevents approximately 50% of the phosphate from being incorporated into FRTL-5 cell thyroglobulin. Approximately one-half of the phosphate in FRTL-5 cell or bovine thyroglobulin can also be released by enzymatic deglycosylation and can be located in Pronase-digested peptides which contain mannose, are endo-beta-N-acetylglucosaminidase H but not neuraminidase-sensitive, and release a dually labeled oligosaccharide containing mannose and phosphate after endo-beta-N-acetylglucosaminidase H digestion. The remainder of the phosphate is in alkali-sensitive phosphoserine residues (3-4/mol of protein) and phosphotyrosine residues (approximately 2/mol of protein). This is evidenced by electrophoresis of acid hydrolysates of 32P-labeled thyroglobulin and by reactivity with antibodies directed against phosphotyrosine residues. The phosphoserine and phosphotyrosine residues do not appear to be randomly located through the thyroglobulin molecule since approximately 75-85% of the phosphotyrosine and phosphoserine residues were recovered in a approximately 15-kDa tryptic peptide or a approximately 24-kDa cyanogen bromide peptide, each almost devoid of carbohydrate. 31P nuclear magnetic resonance studies of bovine thyroglobulin confirm the presence and heterogeneity of the phosphate residues on thyroglobulin preparations.     

Role of the vascular laboratory in the diagnosis of aortic arch occlusive disease: Screening of 2000 patients

A significant difference in bilateral systolic brachial artery pressures and the presence of systolic bruit over a subclavian artery suggests occlusive disease in the innominate or subclavian arteries. Two thousand patients with these signs underwent ultrasonography of the carotid, subclavian, and brachial arteries in our laboratory. Doppler recordings of a vertebral artery, with hyperemia testing of the ipsilateral arm, identified those with a subclavian steal syndrome. In each case, angiography confirmed the laboratory diagnosis. The effectiveness of corrective surgery was evaluated noninvasively. Illustrative cases are presented and discussed.