Transient neonatal hyperthyrotrophinaemia: a serum abnormality due to transplacentally acquired antibody to thyroid stimulating hormone.

In a screening programme for neonatal hypothyroidism an otherwise healthy female infant was found to have a high concentration of thyroid stimulating hormone in a filter paper blood spot and in serum. A high concentration was also found in the maternal serum. Mother and baby were both biochemically euthyroid with normal serum thyroxine concentrations. The apparently high concentration of thyroid stimulating hormone in the mother was due to the presence of an IgG antibody that bound to human but not bovine thyroid stimulating hormone. Maternal serum inhibited the action of human thyroid stimulating hormone in an in vitro bioassay for the hormone. It is suggested that the baby acquired the antibody transplacentally, especially as the concentration of thyroid stimulating hormone subsequently fell. It is concluded that maternal serum should be assayed for thyroid stimulating hormone when a neonate is found to have a high concentration of the hormone and a normal concentration of thyroxine to establish the incidence of this finding and to avoid inappropriate replacement treatment.     

Differing Neurochemical and Morphological Sequelae of Global Ischemia: Comparison of Single- and Multiple-Insult Paradigms

The purpose of this investigation was to investigate pathomechanisms responsible for the deleterious effects of repeated episodes of brief forebrain ischemia. Halothane-anesthetized male Wistar rats were subjected to either (a) a single 15-min period or (b) three 5-min periods (separated by 1 h) of global forebrain ischemia by bilateral carotid artery occlusions plus hypotension (50 mm Hg), followed by various periods of recirculation. Brain temperature was normothermic throughout. In one series of rats, extracellular levels of glutamate, glycine, and gamma-aminobutyric acid (GABA) were measured in the dorsolateral striatum (n = 6-8 per group) and lateral thalamus (n = 4-6 per group) by microdialysis and HPLC before and during ischemia and during 3-5 h of recirculation. In a parallel series of rats (n = 6 per group), ischemic cell change was quantified at 2 (dark neurons), 24, or 72 h following either single or multiple ischemic insults. A single 15-min ischemic period led to massive glutamate release (13-fold increase; p = 0.001), which returned to normal by 20-30 min of recirculation and remained normal thereafter. By contrast, in rats with three 5-min periods of ischemia, the glutamate level rise with each repeated insult (four- to 4.5-fold; p < or = 0.02) was smaller than that observed during the single 15-min insult, but a late sustained rise (five- to six-fold; p < 0.05) occurred at 2-3 h of recirculation. Brief ischemia-induced elevations of glycine and GABA levels were detected in both the single- and multiple-insult groups, with normalization during recirculation. In contrast, the excitotoxic index, a composite measure of neurotransmitter release ([glutamate] x [glycine]/[GABA]), differed markedly following single versus multiple insults (p = 0.002 by repeated-measures analysis of variance) and increased by seven- to 12-fold (p < 0.05) at 1-3 h following the third insult. The total amount of glutamate released was 3.3-fold higher in the multiple-insult than in the single-insult group (p < 0.02). At 2 h of recirculation, histopathological analysis of dorsolateral striatum showed a significantly greater frequency of dark neurons in the multiple- than in the single-insult group (p < 0.05 by analysis of variance). In the thalamus, a higher frequency of ischemic neurons was seen in the multiple-than in the single-insult group at all intervals studied. Thus, in rats with multiple ischemic insults, accelerated ischemic damage was found in the striatum, and severe ischemic injury was documented in the thalamus.(ABSTRACT TRUNCATED AT 400 WORDS)     

Studies of the sites of intracellular degradation of apolipoprotein B in Hep G2 cells.

We previously reported that treatment of Hep G2 cells with oleate significantly increased apolipoprotein B (apoB) secretion by reducing early intracellular degradation of nascent apoB. In the current study, inhibitors of secretory protein transport (brefeldin A and monensin), cell fractionation studies, and protease protection assays were utilized to determine the location of apoB degradation and to better define the mechanism whereby oleate treatment reduces nascent apoB intracellular degradation. When cells were treated with brefeldin A, which blocks endoplasmic reticulum (ER) to Golgi protein transport, apoB degradation continued in control cells, suggesting that apoB is degraded in the ER. When oleate-treated cells were blocked with brefeldin A, oleate failed to protect apoB from intracellular degradation. The effects of brefeldin A were not due to effects on lipid synthesis as brefeldin A did not inhibit the synthesis of triglyceride, phospholipid, free cholesterol, or cholesteryl ester in control cells and did not prevent the increases in triglyceride (14-fold) and phospholipid (1.4-fold) synthesis seen in oleate-treated cells. Simultaneous treatment of cells with brefeldin A and nocodazole, which inhibits retrograde transport of proteins from Golgi to ER, added to the evidence for the ER as the site of apoB degradation. This conclusion received further support from experiments in which cells were treated with monensin, a Na+ ionophore which halts protein secretion at the level of the trans-Golgi network. Early degradation of nascent apoB (between 10 and 20 min of chase) was observed in monensin-treated cells, but then cellular apoB degradation ceased and apoB was stable during the remaining chase period. More apoB accumulated in the Golgi of cells that had been treated with oleate and monensin. These results suggest that ER degradation occurs in monensin-treated cells, but then stops as apoB is transferred to the Golgi. The results obtained in whole cells were confirmed in studies using isolated ER and Golgi, which indicated that ER contains a proteolytic activity which degrades apoB, in vitro, whereas Golgi does not. ApoB degradation in isolated ER was not reduced by pretreatment with oleate. Finally, protease protection assays carried out with isolated microsomes indicated that a majority of the apoB in both control or oleate-treated HepG2 cells was located on the cytosolic side of the membranes.(ABSTRACT TRUNCATED AT 400 WORDS)     

Concanavalin A induces interactions between surface glycoproteins and the platelet cytoskeleton

We have measured the association of platelet surface membrane proteins with Triton X-100 (Triton)-insoluble residues in platelets surface labeled with 125I. In both concanavalin A (Con A)-stimulated and resting platelets, this fraction is composed largely of polypeptides with apparent molecular weights of 45,000, 200,000, and 250,000 which comigrate with authentic actin, myosin heavy chain, and actin binding protein, respectively, as judged by PAGE in SDS. Less than 10% of the two major 125I-labeled surface glycoproteins, GPiib and GPIII, were associated with the Triton residue in resting platelets. Within 45 s after Con A addition, 80-95% of these two glycoproteins became associated with the Triton residue and the amount of sedimentable actin doubled. No cosedimentation of GPIIb and III with the cytoskeletal protein-containing Triton residue was seen when Con A was added to a Triton extract of resting cells, indicating that the sedimentation of GPIIb and III seen in Con A-stimulated platelets was not due to precipitation of the glycoproteins by Con A after detergent lysis. Treatment of Triton extracts of Con A-stimulated platelets with DNase I (deoxyribonucleate 5'-oligonucleotidido-hydrolase [EC 3.1.4.5]) inhibited the sedimentation of actin and the two surface glycoproteins in a dose-dependent manner. This inhibition of cosedimentation was not due to an effect of DNase I on Con A-glycoprotein interactions since these two glycoproteins could be quantitatively recovered by Con A- Sepharose affinity absorption in the presence of DNase I. When the Con A bound to the Triton residue was localized ultrastructurally, it was associated with cell-sized structures containing filamentous material. In intact cells, there was simultaneous immunofluorescent coredistribution of surface-bound Con A and myosin under conditions which induced a redistribution of platelet myosin. These data suggest that Con A can, in the intact platelet, induce physical interactions between certain surface glycoproteins and the internal cytoskeleton.     

Purification and preliminary immunological characterization of the type 5 adenovirus, nonstructural 100,000-dalton protein.

The nonstructural 100,000-dalton (100K) protein of type 5 adenovirus was isolated and purified from infected KB cells by a combination of ion-exchange and affinity chromatographies. Rabbit antiserum containing specific 100K protein antibodies was used for indirect immunofluorescence examination of cells infected with wild-type virus, 100K mutants, and hexon mutants. The 100K protein, which is synthesized as a late protein, was observed primarily in the cytoplasm of cells infected with wild-type and mutant viruses.     

A Substrate for Direct Measurement of L-iduronic Acid 2-sulfate sulfatase

Commercially available sodium heparinate has been sequentially treated with methanolic 0.06M hydrogen chloride and nitrous acid. The nondegraded material was separated by gel filtration from the nonsulfated and monosulfated disaccharides produced. The latter ones, obtained in 10% yield, have been used as a substrate for the direct measurement of the enzyme L-iduronic acid 2-sulfate sulfatase present in human plasma and fibroblast homogenates. Studies of the kinetics and pH optimum of the enzyme, by use of plasma of a patient with mucolipidosis II, indicated an apparent K_m of 2.5mM and a pH optimum of 4.6-4.8. The levels of activity in normal plasma and plasma of a patient with Hunter's disease were found to be 20.4 ± 1.22 units (μmol sulfate/24 h/g protein) and 3.25 ± 0.35 units, respectively. In homogenates of cultured skin fibroblasts, the levels were 137.6 ± 10.7 units for normal controls and 6.4 ± 5.1 for patients with Hunter's disease. The plasma of two obligated heterozygotes gave intermediate levels of activity, whereas the plasma of two possible heterozygotes gave either intermediate levels or entirely normal levels of activity.     

Shifts in ATP synthesis during preimplantation stages of mouse embryos.

The actual and potential activities of the cyochrome system were studied in cleavage-stage mouse embroys. Activities were determined by assaying embroys for total ATP and the rates of [32-P]ATP synthesis both before and after their incubation in medium supplemented either with an energy coupling site inhibitor (antimycin, amytal or cyanide) or with the FADH-linked substrate, succinate. The data indicate that there are three major shifts in the mode of ATP production during preimplantation stages: the first, between the two-cell and late four-cell stages; the second, between the eight-celland late morula stages; and the third, between the late morula and late blastocyst stages. These data are discussed in relation to studies on the energy metabolism of cleavage and blastocyst stage mouse embryos.