Maternal First Trimester TSH Concentrations: Do They Affect Perinatal and Endocrine Outcomes?

We aimed to examine the distribution of 1(st) trimester TSH and evaluate its association with perinatal outcomes and future development of maternal thyrotoxicosis. This retrospective cohort study included data of all women without prior thyroid disease who delivered a singleton at our medical center from 1/2001 to 12/2011 and had a 1(st) trimester TSH<4.0 mU/l. Women were divided according to 1(st) trimester TSH concentrations into quartiles and by predefined TSH values (mU/l): 1) TSH<0.1; 2) TSH 0.11-0.2; 3) TSH 0.21-0.4; and 4) TSH 0.4-4. Obstetrical outcomes, hCG concentrations, and future thyroid status were collected from electronic medical records. A total of 13 841 women fulfilled the inclusion criteria. Mean maternal TSH concentration at 5 weeks of gestation was 2.09±0.83 mU/l and decreased to 1.29±0.87 mU/l in weeks 8-9 with an increase towards the end of the 1(st) trimester. Odds ratio for future thyrotoxicosis was 3.64 in the lowest compared to the highest TSH quartile and 10.03 in those with TSH<0.1 compared to TSH 0.41-4 mU/l. Rates of female fetuses were higher in the low TSH quartiles and in the lower TSH groups, however baby gender was not associated with increased risk of future thyrotoxicosis. Low maternal 1(st) trimester TSH quartiles or concentrations were not associated with adverse pregnancy outcome. Only a minor fraction of pregnant women with a low first tirmester TSH subsequently developed future thyrotoxicosis.

Oral administration of immunoglobulin G-enhanced colostrum alleviates insulin resistance and liver injury and is associated with alterations in natural killer T cells.

Insulin resistance and metabolic syndrome are chronic inflammatory conditions that lead to hepatic injury and non-alcoholic steatohepatitis (NASH). Bovine colostrum has therapeutic effects in a variety of chronic infections. However its effectiveness in NASH was never studied. Natural killer T (NKT) cells have been shown to be associated with some of the pathological and metabolic abnormalities accompanying NASH in leptin-deficient (ob/ob) mice. In the present study, we used hyperimmune bovine colostrum to treat hepatic injury and insulin resistance and we also assessed the effects on NKT cells. We used ob/ob mice that were fed for 6 weeks with either 0·1 mg bovine colostrum prepared from non-immunized cows, 0·1 mg hyperimmune colostrum raised against a bacterial lipopolysaccharide (LPS) extract or 0·001, 0·1 or 1 mg of immunoglobulin (Ig)G purified from hyperimmune colostrum (IgG-LPS). NKT cells were phenotyped by flow cytometry, and hepatic injury and insulin resistance were assessed by measuring fasting glucose levels, glucose tolerance tests and liver enzymes. Fat accumulation was measured in the liver and plasma. Oral administration of hyperimmune colostrums decreased alanine aminotransferase (ALT) serum levels and serum triglycerides compared to controls. Glucose intolerance was also improved by the hyperimmune colostrum preparations. These results were accompanied by a decrease in serum tumour necrosis factor (TNF)-α levels following oral treatment with 0·1 or 1 mg of IgG-LPS. The beneficial effects of hyperimmune colostrums were associated with an increase in the number of splenic NKT cells. These data suggest that oral administration of hyperimmune colostrum preparations can alleviate chronic inflammation, liver injury and insulin resistance associated with NASH.

Cyclosporin A-dependent downregulation of the Na+/Ca2+ exchanger expression.

Cyclosporin A (CsA) is an immunosuppressive drug commonly given to transplant patients. Its application is accompanied by severe side effects related to calcium, among them hypertension and nephrotoxicity. The Na+/Ca2+ exchanger (NCX) is a major calcium regulator expressed in the surface membrane of all excitable and many nonexcitable tissues. Three genes, NCX1, NCX2, and NCX3 code for Na+/Ca2+ exchange activity. NCX1 gene products are the most abundant. We have shown previously that exposure of NCX1-transfected HEK 293 cells to CsA, leads to concentration-dependent reduction of Na+/Ca2+ exchange activity and surface expression, without a reduction in total cell-expressed NCX1 protein. We show now that the effect of CsA on NCX1 protein expression is not restricted to transfected cells overexpressing the NCX1 protein but exhibited also in cells expressing endogenously the NCX1 protein (L6, H9c2, and primary smooth muscle cells). Exposure of NCX2- and NCX3-transfected cells to CsA results also in reduction of Na+/Ca2+ exchange activity and surface expression, though the sensitivity to the drug was lower than in NCX1-transfected cells. Studying the molecular mechanism of CsA-NCX interaction suggests that cyclophilin (Cyp) is involved in NCX1 protein expression and its modulation by CsA. Deletion of 426 amino acids from the large cytoplasmic loop of the protein retains the CsA-dependent downregulation of the truncated NCX1 suggesting that CsA-Cyp-NCX interaction involves the remaining protein domains.

Rab4 regulates formation of synaptic-like microvesicles from early endosomes in PC12 cells.

Early endosomes in PC12 cells are an important site for the formation of synaptic-like microvesicles and constitutive recycling vesicles. By immunogold electron microscopy, the small GTPase rab4 was localized to early endosomes and numerous small vesicles in the cell periphery and Golgi area of PC12 cells. Overexpression of GTPase-deficient Q67Lrab4 increased the number of early endosome-associated and cytoplasmic vesicles, whereas expression of GDP-bound S22Nrab4 significantly increased the length of early endosomal tubules. In parallel, Q67Lrab4 induced a shift in rab4, VAMP2, and TfR label from early endosomes to peripheral vesicles, whereas S22Nrab4 increased early endosome labeling of all three proteins. These observations were corroborated by early endosome budding assays. Together, our data document a thus far unrecognized role for rab4 in the formation of synaptic-like microvesicles and add to our understanding of the formation of constitutive recycling vesicles from early endosomes.

Synaptic vesicles form by budding from tubular extensions of sorting endosomes in PC12 cells.

The putative role of sorting early endosomes (EEs) in synaptic-like microvesicle (SLMV) formation in the neuroendocrine PC12 cell line was investigated by quantitative immunoelectron microscopy. By BSA-gold internalization kinetics, four distinct endosomal subcompartments were distinguished: primary endocytic vesicles, EEs, late endosomes, and lysosomes. As in other cells, EEs consisted of vacuolar and tubulovesicular subdomains. The SLMV marker proteins synaptophysin and vesicle-associated membrane protein 2 (VAMP-2) localized to both the EE vacuoles and associated tubulovesicles. Quantitative analysis showed that the transferrin receptor and SLMV proteins colocalized to a significantly higher degree in primary endocytic vesicles then in EE-associated tubulovesicles. By incubating PC12 cells expressing T antigen-tagged VAMP (VAMP-TAg) with antibodies against the luminal TAg, the recycling pathway of SLMV proteins was directly visualized. At 15 degrees C, internalized VAMP-TAg accumulated in the vacuolar domain of EEs. Upon rewarming to 37 degrees C, the labeling shifted to the tubular part of EEs and to newly formed SLMVs. Our data delineate a pathway in which SLMV proteins together with transferrin receptor are delivered to EEs, where they are sorted into SLMVs and recycling vesicles, respectively.

Reconstitution of synaptic vesicle biogenesis from PC12 cell membranes.

Neuroendocrine PC12 cells contain small microvesicles that closely resemble synaptic vesicles in their physical and chemical properties. Two defining characteristics of synaptic vesicles are their homogeneous size and their unique protein composition. Since synaptic vesicles arise by endocytosis from the plasma membrane, nerve terminals and PC12 cells must contain the molecular machinery to sort synaptic vesicles from other membrane proteins and pinch off vesicles of the correct diameter from a precursor compartment. A cell-free reconstitution system was developed that generates vesicles from PC12 membrane precursors in the presence of ATP and brain cytosol and is temperature dependent. At 15 degrees C, surface-labeled synaptic vesicle proteins accumulate in a donor compartment, while labeled synaptic vesicles cannot be detected. The block of synaptic vesicle formation at 15 degrees C enables the use of the monoclonal antibody, KT3, a specific marker for the epitope-tagged synaptic vesicle protein, VAMP-TAg, to label precursors in the synaptic vesicle biogenesis pathway. From membranes labeled in vivo at 15 degrees C, vesicles generated in vitro at 37 degreesC had the sedimentation characteristics of neuroendocrine synaptic vesicles on glycerol velocity gradients, and excluded the transferrin receptor. Therefore, vesiculation and sorting can be studied in this cell-free system.

Vesiculation and sorting from PC12-derived endosomes in vitro.

Formation of small vesicles resembling synaptic vesicles can be reconstituted in vitro by incubating labeled homogenates of PC12 cells with ATP and two cytoplasmic proteins, AP3 and ARF1 [Faúndez, V., Horng, J.-T. & Kelly, R. B. (1998) Cell 93, 423-432]. To determine whether AP3 was mediating budding from plasma membranes or endosomes the organelle that generated the synaptic vesicles was characterized. The budding activity was enriched in organelles that labeled at 15 degrees C, but not at 4 degrees C, that excluded a marker of plasma membranes and that contained internalized transferrin, indicating that the precursor was an endosome. Vesicles formed from the endosomal precursor in vitro excluded transferrin. We conclude that ARF-mediated vesiculation into synaptic vesicle-sized organelles uses an endosomal precursor and occurs simultaneously in vitro with sorting of synaptic vesicle proteins from other membrane protein constituents of the endosome.

Normal and atypical butyrylcholinesterases in placental development, function, and malfunction.

1. In utero exposure to poisons and drugs (e.g., anticholinesterases, cocaine) is frequently associated with spontaneous absorption and placental malfunction. The major protein interacting with these compounds is butyrylcholinesterase (BuChE), which attenuates the effects of such xenobiotics by their hydrolysis or sequestration. Therefore, we studied BuChE expression during placental development. 2. RT-PCR revealed both BuChEmRNA and acetylcholinesterase (AChE) mRNA throughout gestation. However, cytochemical staining detected primarily BuChE activity in first-trimester placenta but AChE activity in term placenta. 3. As the atypical variant of BuChE has a narrower specificity for substrates and inhibitors than the normal enzyme, we investigated its interactions with alpha-solanine and cocaine, and sought a correlation between the occurrence of this variant and placental malfunction. 4. Atypical BuChE of serum or recombinant origin presented > 10-fold weaker affinities than normal BuChE for cocaine and alpha-solanine. However, BuChE in the serum of the heterozygote and a homozygous normal were similar in their drug affinities. Therefore, heterozygous serum or placenta can protect the fetus from drug or poison exposure, unlike homozygous atypical serum or placenta. 5. Genotype analyses revealed that heterozygous carriers of atypical BuChE were threefold less frequent among 49 patients with placental malfunction than among 76 controls of the entire Israeli population. These observations exclude heterozygote carriers of atypical BuChE from being at high risk for placental malfunction under exposure to anticholinesterases.

Overlapping drug interaction sites of human butyrylcholinesterase dissected by site-directed mutagenesis.

Butyrylcholinesterase [BuChE (acylcholine acyl hydrolase); EC 3.1.1.8] limits the access of drugs, including tacrine, to other proteins. The "atypical" BuChE variant, in which Asp70 at the rim of the active site gorge is substituted by glycine, displayed a more drastically weakened interaction with tacrine than with cocaine, dibucaine, succinylcholine, BW284c51 [1,5-bis(4-allyldimethylammoniumphenyl)pentan-3-one dibromide], or alpha-solanine. To delineate the protein domains that are responsible for this phenomenon, we mutated residues within the rim of the active site gorge, the region parallel to the peripheral site in the homologous enzyme acetylcholinesterase [AChE (acetylcholine acetyl hydrolase); EC 3.1.1.7], the oxyanion hole, and the choline-binding site. When expressed in microinjected Xenopus laevis oocytes, all mutant DNAs yielded comparable amounts of immunoreactive protein products. Most mutants retained catalytic activity close to that of wild-type BuChE and were capable of binding ligands. However, certain modifications in and around the oxyanion hole caused a dramatic loss in activity. The affinities for tacrine were reduced more dramatically than for all other ligands, including cocaine, in both oxyanion hole and choline-binding site mutants. Modified ligand affinities further demonstrated a peripheral site in residues homologous with those of AChE. BuChE mutations that prevented tacrine interactions also hampered its ability to bind other drugs and inhibitors, which suggests a partial overlap of the binding sites. This predicts that in addition to their genetic predisposition to adverse responses to tacrine, homozygous carriers of "atypical" BuChE will be overly sensitive to additional anticholinesterases and especially so when exposed to several anticholinesterases in combination.

Genetic predisposition to adverse consequences of anti-cholinesterases in 'atypical' BCHE carriers.

Normal butyrylcholinesterase (BuChE), but not several of its common genetic variants, serves as a scavenger for certain anti-cholinesterases (anti-ChEs). Consideration of this phenomenon becomes urgent in view of the large-scale prophylactic use of the anti-ChE, pyridostigmine, during the 1991 Persian Gulf War, in anticipation of nerve gas attack and of the anti-ChE, tacrine, for improving residual cholinergic neurotransmission in Alzheimer's disease patients. Adverse symptoms were reported for subjects in both groups, but have not been attributed to specific causes. Here, we report on an Israeli soldier, homozygous for 'atypical' BuChE, who suffered severe symptoms following pyridostigmine prophylaxis during the Persian Gulf War. His serum BuChE and recombinant 'atypical' BuChE were far less sensitive than normal BuChE to inhibition by pyridostigmine and several other carbamate anti-ChEs. Moreover, atypical BuChE demonstrated 1/200th the affinity for tacrine of normal BuChE or the related enzyme acetylcholinesterase (AChE). Genetic differences among BuChE variants may thus explain at least some of the adverse responses to anti-ChE therapies.

Successive organophosphate inhibition and oxime reactivation reveals distinct responses of recombinant human cholinesterase variants.

To explore the molecular basis of the biochemical differences among acetylcholinesterase (AChE), butyrylcholinesterase (BuChE) and their alternative splicing and allelic variants, we investigated the acylation phase of cholinesterase catalysis, using phosphorylation as an analogous reaction. Rate constants for organophosphate (DFP) inactivation, as well as for oxime (PAM)-promoted reactivation, were calculated for antibody-immobilized human cholinesterases produced in Xenopus oocytes from natural and site-directed variants of the corresponding DNA constructs. BuChE displayed inactivation and reactivation rates 200- and 25-fold higher than either product of 3'-variable AChE DNAs, consistent with a putative in vivo function for BuChE as a detoxifier that protects AChE from inactivation. Chimeric substitution of active site gorge-lining residues in BuChE with the more anionic and aromatic residues of AChE, reduced inactivation 60-fold but reactivation only 4-fold, and the rate-limiting step of its catalysis appeared to be deacylation. In contrast, a positive charge at the acyl-binding site of BuChE decreased inactivation 8-fold and reactivation 30-fold. Finally, substitution of Asp70 by glycine, as in the natural 'atypical' BuChE variant, did not change the inactivation rate yet reduced reactivation 4-fold. Thus, a combination of electrostatic active site charges with aromatic residue differences at the gorge lining can explain the biochemical distinction between AChE and BuChE. Also, gorge-lining residues, including Asp70, appear to affect the deacylation step of catalysis by BuChE. Individuals carrying the 'atypical' BuChE allele may hence be unresponsive to oxime reactivation therapy following organophosphate poisoning.