The human thyrotropin (TSH) receptor in a TSH binding inhibition assay for TSH receptor autoantibodies

Seven years after the molecular cloning of the human TSH receptor (TSHR), the porcine TSHR remains in general use in the TSH binding inhibition (TBI) assay for autoantibodies to the TSHR. We compared porcine and recombinant human TSHR in two types of TBI assays: one using intact Chinese hamster ovary cells expressing the recombinant human TSHR on their surface, and the other using soluble receptors extracted from these cells with detergent. In the intact cell TBI assay, monolayers expressing large numbers of TSHR were less effective than cells expressing few receptors. These findings are consistent with the very low concentration of TSHR autoantibodies in serum. Binding of [125I]human TSH was about 5-fold lower than that of [125I]bovine TSH to the intact cells. Nevertheless, TBI values with the two ligands were similar for most sera. However, a few sera produced greater inhibition of human than of bovine TSH binding. In the solubilized human TSHR TBI assay, in contrast to the intact cell TBI assay, cells expressing very large number of TSHR were an excellent source for detergent extraction of soluble human TSHR, but only if the cells were extracted while still on the dish and not after scraping. A 10-cm diameter dish of cells provided TSHR for 100-200 replicate determinations when substituted for solubilized porcine TSHR in a commercial TBI kit. TBI values in serum from 30 individuals with suspected Graves' disease correlated closely when tested with solubilized human and porcine TSHR (r = 0.954; P < 0.001). However, 2 sera that were negative with the porcine TSHR were positive with the human TSHR. TBI and thyroid-stimulating activity in these sera correlated weakly regardless of whether the TBI used human or porcine TSHR. These findings open the way to a practical TBI assay using recombinant human TSHR.     

IL-1/IL-3 gene therapy of non-small cell lung cancer (NSCLC) in rats using 'cracked' adenoproducer cells

Cytokine gene therapy was studied in established L42 tumours in syngeneic rats. L42 is a transplantable non-immunogenic non-small cell lung cancer (NSCLC). Genes coding for human interleukin-1 alpha and for rat interleukin-3 beta were transferred by injecting producer cells of recombinant adenovirus vectors into the tumour in attempts to achieve high concentrations of the cytokines inside the tumor without systemic toxicity. Limited tumour growth delay was obtained with viable producer cells. For logistic reasons stocks of pooled frozen producer cells allowed intensive treatment of groups of tumour bearing rats. The cells were lysed by thawing before administration. Ten daily injections of such 'cracked' producer cells induced reproducible tumour responses. These were due to local release of cytokines, not to systemic effects. Growth retardation also occurred in contralateral tumours which were not injected. When rats carrying established tumours were vaccinated with lysates of tumours collected during treatment with 'cracked' producer cells, significant tumour growth retardation was obtained. We speculate that both cytokines, if produced at sufficiently high concentrations in tumours, induce inflammation which in turn initiates an immune response against tumours growing at a distant site. These findings seem to justify further exploration of IL-1 and IL-3 gene transfer for the treatment of cancers.     

Determination of a specific 5-HT2a antagonist (M100907) in rat and dog plasma at the femtomole/milliliter level by gas chromatography-mass spectrometry

Hypothyroidism was induced in a group of male Fischer 344 rats by administration of 0.05% propylthiouracil (PTU) in the drinking water for 12 weeks. Control rats were not treated. Plasma levels of thyroid hormones indicated that PTU treatment had produced severe thyroid hormone deficiency. In PTU-treated rats compared to control rats, levels of total T3 and total T4 were reduced 54.5% and 53.7%; while levels of free T3 and free T4 were reduced 87.1% and 96.5%. Functional hypothyroidism was demonstrated by: (i) a 49.1% decrease in hepatic plasma membrane alpha1-adrenergic receptor binding, and (ii) a 11.2-fold increase in hepatic gamma-glutamyltranspeptidase activity; relative to the expression of these parameters in control rats. Membranes were isolated from hippocampi of control, PTU-induced hypothyroid and thyroxine-replaced rats and specific adrenergic receptor binding determined by radioligand binding techniques. Hypothyroidism resulted in a shift in the balance of alpha1 and beta2 adrenergic receptor binding by evoking: an increase in alpha1-adrenergic receptor binding to 1.57-fold of control levels; and, a decrease in beta2-adrenergic receptor binding to 64% of control levels. Thyroid hormone replacement carried out in PTU-treated hypothyroid rats at 30 microg/kg s.c. per day for the last 3 days of the 12 week PTU-treatment protocol, which reversed physical and functional hypothyroidism, reversed the observed changes in hippocampal adrenergic receptor binding, indicating them to be thyroid hormone, and not PTU, -dependent. This receptor shift evoked by hypothyroidism may, in part, explain the protective effect of hypothyroidism on ischemia-induced hippocampal damage by favoring inhibitory input and limiting excitotoxic input by catecholamines.     

A relaxin-mediated pathway to preterm premature rupture of the fetal membranes that is independent of infection

The MAP kinase pathway has been shown to be active in many growth factor signaling systems, including that of prolactin (PRL). In our studies, the main objective was to examine the possible involvement of MEK kinases (Map/Erk kinase kinases) in PRL-stimulated mitogenic and lactogenic processes. We used the MEK kinase inhibitor PD 098059 to block MEK kinase activation in the Nb2 cell line and mammary gland explants derived from 12- to 14-day pregnancy mice. PD 098059 attenuated PRL-induced Nb2 cell mitogenesis at 10 microM and a maximum inhibition was observed at 100 microM. In cultured mammary tissues, PD 098059 at 100 microM had no effect on the PRL stimulation of lipid, casein and lactose synthesis and iodide uptake. Further, the growth-inhibitory effect of PD 098059 on Nb2 cells was ameliorated when the drug was removed from the culture medium, indicating that PD 098059 acts in a reversible manner. When MEK1 was immunoprecipitated from PD 098059 and/or PRL treated Nb2 cells, PRL-stimulated MEK1 kinase activity was directly inhibited by PD 098059 at concentrations employed in the culture experiments. PRL has no effect on the tyrosyl phosphorylation of MAP kinases in cultured mammary tissues derived from pregnant mice, whereas earlier we found that PRL stimulates the tyrosyl phosphorylation of all four MAP kinases in Nb2 cells. The results suggest that the MAP kinase pathway plays an important role in the PRL stimulation of Nb2 cell mitogenesis but is not involved in the PRL stimulation of milk product synthesis.     

Growth Kinetics of Mixed Cultures under Chlorate-Reducing Conditions

Although chlorate (ClO3?) is not a naturally occurring compound, chlorate can serve as an electron acceptor for a number of microorganisms. To investigate the rate that chlorate-reducing microbes can grow on different substrates, growth rate constants were determined in chemostat studies under chlorate-reducing conditions. Maximum growth rates using acetate, glucose-glutamic acid, and phenol were 0.56, 0.12, and 0.040 h?1, with cell yields of 0.12, 0.41, and 0.12 g-cell∕g-substrate, respectively.     

Functional expression of the apical Na+-dependent bile acid transporter in large but not small rat cholangiocytes

BACKGROUND & AIMS: Bile acids interact with cholangiocytes, resulting in cholangiocyte proliferation and increases in ductal bile secretion in large but not small cholangiocytes. It was proposed that for bile acids to exert these effects on cholangiocytes, a specific uptake mechanism must be present in cholangiocytes. The aim of this study was to show the expression of a bile acid transporter in cholangiocytes. METHODS: Small and large cholangiocytes or intrahepatic bile duct units (IBDUs) were isolated from normal rats, and gene expression for the apical Na+-dependent bile acid transporter (ABAT) and the 14-kilodalton ileal cytosolic binding protein (IBABP) was assessed by ribonuclease-protection assays. Tissue and subcellular distribution of bile acid transporters was also studied. [14C]-Taurocholate uptake into cholangiocytes was determined. RESULTS: Both ABAT and IBABP messenger RNAs were detected in large but not small cholangiocytes. By immunohistochemistry, ABAT was present in large but not small cholangiocytes. Immunofluorescence showed ABAT to be present in the apical membrane of large IBDUs. A Na+-dependent saturable uptake of taurocholate was present in large but not small cholangiocytes. CONCLUSIONS: These proteins may mediate bile acid uptake from the duct lumen in large ducts, resulting in modification of canalicular bile secretion and modulation of ductal bile secretion and growth.     

A CaMV 35S promoter driven cDNA clone of tobacco mosaic virus can infect host plant tissue despite being uninfectious when manually inoculated onto leaves

The amino acid and carbohydrate analysis of scyllin, a low molecular weight lectin purified from Scylla serrata (edible crab) haemolymph reveal that scyllin is rich in acidic and neutral amino acids and contains high amount of mannose. UV absorption of scyllin is perturbed by DMSO at 272 nm showing the presence of tryptophan molecule in scyllin exposed and accessible to the solvent. The oxidation of tryptophan molecule by N-bromosuccinimide results in loss of haemagglutinating activity of lectin. The study of thermodynamic parameters of scyllin-glycoproteins interaction suggests that ceruloplasmin is the most potent inhibitors of scyllin of all the glycoproteins studied.     

Modeling unfolded states of proteins and peptides. II. Backbone solvent accessibility

Buried surface area is often used as a measure of the contribution to protein folding from the hydrophobic effect. Quantitatively, the surface buried upon folding is reckoned as the difference in area between the native and unfolded states. This calculation is well defined for a known structure but model-dependent for the unfolded state. In a previous paper [Creamer, T. P., Srinivasan, R., & Rose, G. D. (1995) Biochemistry 34, 16245-16250], we developed two models that bracket the surface area of the unfolded state between limiting extremes. Using these extrema, it was shown that earlier models, such as an extended tripeptide, overestimate the surface area of side chains in the unfolded state. In this sequel to our previous paper, we focus on backbone surface in the unfolded state, again adopting the strategy of trapping the area between limiting extrema. A principal conclusion of this present study is that most backbone surface in proteins is buried within local structure.     

Advances in the genetic mechanisms of mitochondrial disease

During the past 16 years since the delineation of the human mitochondrial genome, substantial advances have been made in identifying pathogenic mutations causing mitochondrial disorders. However, just as we have come to accept the unexpected in the nontraditional aspects of Mendelian inheritance with the discovery of trinucleotide expansions, imprinting and uniparental disomy, unusual characteristics of mitochondrial inheritance also have been found that defy existing laws. For example, we now know that the nuclear genetic background of an individual might influence the expression and tissue specificity of mitochondrial mutations. Pathogenic mitochondrial DNA mutations contribute to the generation of new mutations by compromising mitochondrial function and increasing free radical production. Evidence for recombination raises new questions about repair mechanisms of mitochondrial DNA. It appears that the more we learn about the bases of mitochondrial disease, the more complex diagnosis, treatment, and genetic counseling become.     

Cloning of the rainbow trout (Oncorhynchus mykiss) Mx2 and Mx3 cDNAs and characterization of trout Mx protein expression in salmon cells

Two rainbow trout (Oncorhynchus mykiss) Mx cDNAs were cloned by using RACE (rapid amplification of cDNA ends) PCR and were designated RBTMx2 and RBTMx3. The deduced RBTMx2 and RBTMx3 proteins were 636 and 623 amino acids in length with molecular masses of 72 and 70.8 kDa, respectively. These proteins, along with the previously described RBTMx1 protein (G. D. Trobridge and J. A. Leong, J. Interferon Cytokine Res. 15:691-702, 1995), have between 88.7 and 96.6% identity at the amino acid level. All three proteins contain the tripartite GTP binding domain and leucine zipper motif common to Mx proteins. A monospecific polyclonal antiserum to an Escherichia coli-expressed fragment of RBTMx3 was generated, and that reagent was found to react with all three rainbow trout Mx proteins. Subsequently, endogenous Mx production in RTG-2 cells induced with poly(IC) double-stranded RNA was detected by immunoblot analysis. The cellular localization of the rainbow trout proteins was determined by transient expression of the RBTMx cDNAs in CHSE-214 (chinook salmon embryo) cells. A single-cell transient-transfection assay was used to examine the ability of each Mx cDNA clone to inhibit replication of the fish rhabdovirus infectious hematopoietic necrosis virus (IHNV). No significant inhibition in the accumulation of the IHNV nucleoprotein was observed in cells expressing either trout Mx1, Mx2, or Mx3 in transiently transfected cells.