[Non-Small Cell Lung Cancer - Development of Parallel Mechanisms of Resistance]. / Nicht kleinzelliges Lungenkarzinom ­ doppelte Resistenzentwicklung.

Acquired resistances to tyrosine kinase inhibitors in non-small cell lung cancer develop after 9 - 12 month. In 60 % of the cases these resistances arise because of a secondary EGFR-T790 M resistance mutation. This report is describing the case of a patient who developed parallel two different mechanisms of resistance: A T790 M resistance mutation and a transformation into a small cell neuroendocrine cancer. Under therapy with Osimertinib and chemotherapy with carboplatin and etoposide the tumor responsed partially.

[What is certain in the treatment of glomerulonephritis?] / Was ist gesichert in der Therapie der Glomerulonephritis?

Glomerulonephritides essentially all belong to the rare diseases; however, they are the most common cause of end-stage renal disease in young adults. Besides obtaining a specific diagnosis via a renal biopsy, assessing the prognosis constitutes the other essential step in the work-up, since this enables a decision to be made on whether supportive care with relatively few adverse effects is sufficient or whether additional immunosuppressive therapy is required. The latter is discussed focusing on the most common European types of glomerulonephritis: immunoglobulin A nephropathy, membranous glomerulonephritis, minimal change nephropathy and focal segmental glomerulosclerosis.

Designing the topology of ion nano-channels in the mesophases of amphiphilic wedge-shaped molecules.

The wedge-shaped amphiphiles bearing sulfonate groups at the tip of the wedge are prone to form ion nano-channels upon exposure to a humid atmosphere. During swelling, water molecules preferentially accumulate in polar regions of the system resulting in the formation of a lyotropic phase. In this work, the details of the structure formation processes occurring upon swelling in water vapour, including determination of the size and topology of the ion nano-channels, are explored. The electron density profiles across the channel are obtained from the fits of the X-ray scattering data with two- and three-phase structural models the applicability of which is critically analysed. The results show that the ion channel size correlates not only with water uptake but also with the molecular architecture such as the structure of the rigid molecular fragment bearing a polar group. These findings can help optimising the ion transport for development of ion-selective membranes.

R-CHOEP-14 improves overall survival in young high-risk patients with diffuse large B-cell lymphoma compared with R-CHOP-14. A population-based investigation from the Danish Lymphoma Group.

BACKGROUND: Optimal treatment of young patients with high-risk diffuse large B-cell lymphoma (DLBCL) remains a matter of debate and requires improvement. The combination chemotherapy with cyclophosphamide, doxorubicin, vincristine and prednisone (CHOP) with addition of etoposide (CHOEP) has in other patient groups been shown to be effective. Further improvement has been accomplished with the use of rituximab in combination with the regimens every 2 weeks (R-CHOP-14, R-CHOEP-14). The aim of the present retrospective population-based study was to compare R-CHOP-14 with R-CHOEP-14 in a cohort of high-risk patients aged 18-60 years with two or more risk factors (stage III-IV, elevated lactate dehydrogenase levels, performance status 2-4). To our knowledge, this is the first study comparing these two regimens in this patient group. METHODS: We obtained data for the period 2004-2009 from the Danish Lymphoma Database. One hundred and fifty-nine patients were eligible to enter the study. Primary end point was overall survival (OS) and secondary end points were response to treatment, progression-free survival (PFS) and safety. RESULTS: Four-year OS was superior in the R-CHOEP-14 group: 75% compared with 62% for R-CHOP-14 (P=0.04). This superiority was also seen for PFS: 4-year PFS was 70% for the R-CHOEP-14 group compared with 58% for the R-CHOP-14 group (P=0.02). CONCLUSION: R-CHOEP-14 is a promising regimen for young patients with high-risk DLBCL with improved OS and PFS compared with R-CHOP-14.

Adoptive immunotherapy for cancer: the next generation of gene-engineered immune cells.

Adoptive cellular immunotherapy involving transfer of tumor-reactive T cells has shown some notable antitumor responses in a minority of cancer patients. In particular, transfer of tumor-infiltrating lymphocytes has resulted in long-term objective responses in patients with advanced melanoma. However, the inability to isolate sufficient numbers of tumor-specific T cells from most malignancies has restricted the broad utility of this approach. An emerging approach to circumvent this limitation involves the genetic modification of effector cells with T cell receptor (TCR) transgenes or chimeric single-chain variable fragment (scFv) receptors that can specifically redirect T cells to tumor. There has been much progress in the design of TCR and scFv receptors to enhance the antigen-specific activation of effector cells and their trafficking and persistence in vivo. Considerable effort has been directed toward improving the safety of this approach and reducing the immunogenicity of the receptor. This review discusses the latest developments in the field of adoptive immunotherapy using genetically modified immune cells that have been transduced with either TCR or scFv receptor transgenes and used in preclinical and clinical settings as anticancer agents.

Neurocognitive performance during acute THC intoxication in heavy and occasional cannabis users.

Performance impairment during Delta(9)-tetrahydrocannabinol (THC) intoxication has been well described in occasional cannabis users. It is less clear whether tolerance develops to the impairing effects of THC in heavy users of cannabis. The aim of the present study was to assess neurocognitive performance during acute THC intoxication in occasional and heavy users. Twenty-four subjects (12 occasional cannabis users and 12 heavy cannabis users) participated in a double-blind, placebo-controlled, two-way mixed model design. Both groups received single doses of THC placebo and 500 microg/kg THC by smoking. Performance tests were conducted at regular intervals between 0 and 8 h after smoking, and included measures of perceptual motor control (critical tracking task), dual task processing (divided attention task), motor inhibition (stop signal task) and cognition (Tower of London). THC significantly impaired performance of occasional cannabis users on critical tracking, divided attention and the stop signal task. THC did not affect the performance of heavy cannabis users except in the stop signal task, i.e. stop reaction time increased, particularly at high THC concentrations. Group comparisons of overall performance in occasional and heavy users did not reveal any persistent performance differences due to residual THC in heavy users. These data indicate that cannabis use history strongly determines the behavioural response to single doses of THC.

Somatostatin inhibits rat hepatic T4-5'-deiodinase. The effect is independent of the associated hypoinsulinemia.

Somatostatin decreases the serum 3,5,3'-triiodothyronine (T3) concentration in athyreotic subjects treated with L-thyroxine (T4). The present study was performed to determine the effect of somatostatin on T4-5'-deiodinase activity in rat tissue homogenate preparations. This enzyme is an important regulator of T3 production. Continuous somatostatin infusion at high dose (4 micrograms/kg per min subcutaneously) and low dose (0.8 micrograms/kg per min subcutaneously) for 48-72 h significantly increased (P less than 0.001) the mean aorta plasma somatostatin-like immunoreactivity concentration to 786 +/- 65 and 448 +/- 58 pg/ml, respectively compared with the normal mean of 69 +/- 17 pg/ml in the carbohydrate-fed rat (20% glucose in water ad lib.). The mean hepatic T4-5'-deiodinase activity at both 48 h (100 +/- 5 pmol/min per 100 mg protein) and 72 h (90 +/- 7 pmol/min per 100 mg protein) was significantly reduced in the high-dose group (P less than 0.005), compared with the mean enzyme activity in the glucose-fed control group (138 +/- 6 pmol/min per 100 mg protein). There was a negative correlation (r = -0.9, P less than 0.01) between the alterations in the peripheral plasma somatostatin-like immunoreactivity concentration and hepatic T4-5'-deiodinase activity. High-dose somatostatin did not consistently lower the serum T3 concentration in the glucose-fed rat. Somatostatin had no effect on pituitary T4-5'-deiodinase activity in the glucose-fed rat. High-dose somatostatin also significantly inhibited (P less than 0.01) the glucose-refeeding reactivation of hepatic T4-5'-deiodinase in the 72-h-fasted rat. The mean enzyme activity after 96 h was 96 +/- 8 pmol/min per 100 mg protein compared with 127 +/- 4 pmol/min per 100 mg protein in the refed control group. Somatostatin had a similar inhibitory effect on serum T3. There was a positive correlation (r = 0.5, P less than 0.01) between the somatostatin-induced alterations in serum T3 and hepatic T4-5'-deiodinase during refeeding. A significant positive correlation (r = 07, P less than 0.005) was noted between the somatostatin effect on hepatic T4-5'-deiodinase activity and the induced hypoinsulinemia in the fed group. In addition, a significant negative correlation (r = -0.9, P less than 0.001) was noted between the suppressed enzyme activity and the serum glucose/insulin ratio in the refed group. However, although low-dose somatostatin also induced the same degree of hypoinsulinemia (P less than 0.05) in the fed and refed groups it had no effect on hepatic T4-5'-deiodinase activity. Furthermore, despite the induction of hyperinsulinemia during refeeding, the high dose somatostatin inhibitory effect on enzyme activity persisted. Thus, somatostatin inhibited hepatic T4-5'-deiodinase activity in the carbohydrate-fed rat and prevented the carbohydrate-refeeding normalization of enzyme activity in the 72-h-fasted rat. The effect of somatostatin on enzyme activity was independent of the associated hypoinsulinemia. In the carbohydrate-fed animal the somatostatin effect was selective, as the hormone had no effect on pituitary T4-5'-deiodinase activity. These data suggest that somatostatin could play a role in the peripheral metabolism of thyroid hormones.

Dietary modification of thyroxine deiodination in rat liver is not mediated by hepatic sulfhydryls.

The enzymatic deiodination of thyroxine (T(4)) is thiol dependent. Fasting (72 h) depresses hepatic T(4) deiodination and lowers the hepatic content of nonprotein sulfhydryls (NP-SH) and reduced glutathione (GSH). It has been proposed that the fasting effect may be mediated through these alterations in hepatic sulfhydryls. To test the importance of tissue (hepatic) thiol content in the modification of T(4) deiodination consequent to dietary manipulation, we examined the sequential deiodination of T(4) to 3,5,3'-triiodothyronine (T(3)) (5'-deiodination) and 3,3',5-triiodothyronine (reverse T(3), rT(3)) (5-deiodination) in liver homogenates without added thiol from groups of rats fed Purina lab chow (P) (a protein-rich diet), glucose alone (G), or glucose plus cysteine (G(c)) for 72 h or fasted (F) for the same period. The initial rate of each reaction was compared to the tissue concentrations of NP-SH and GSH. Dietary manipulation induced significant changes in hepatic deiodination of T(4) to T(3) and rT(3) and sulfhydryl content. There was a marked dissociation between the rate of each reaction and hepatic NP-SH and GSH levels. T(4) deiodination by the alternative pathways was significantly higher (P < 0.01) in G > P > F. In contrast both hepatic NP-SH and GSH concentrations were greater (P < 0.05) in P > F > G. The lack of a relationship between these parameters was further emphasized on analysis of tissue from rats fed G(c). Despite the clearcut (P < 0.01) increase in hepatic NP-SH and GSH consequent to G(c) feeding, there was no alteration in iodothyronine deiodination compared to the group fed glucose alone. These data indicate that the effects of diet on T(4) monodeiodination in liver are not mediated by changes in the tissue level of sulfhydryl compounds but rather involve alterations in the concentrations of the deiodinases.

Antitumor activity of dual-specific T cells and influenza virus.

Activation and expansion of T cells are important in disease resolution, but tumors do not usually satisfy these immune requirements. Therefore, we employed a novel strategy whereby dual-specific T cells were generated that could respond to both tumor and influenza virus, reasoning that immunization with influenza virus would activate and expand tumor-specific cells, and inhibit tumor growth. Dual-specific T cells were generated by gene modification of influenza virus-specific mouse T cells with a chimeric gene-encoding reactivity against the erbB2 tumor-associated antigen. Dual-specific T cells were demonstrated to respond against both tumor and influenza in vitro, and expanded in vitro in response to influenza to a much greater degree than in response to tumor cells. Following adoptive transfer and immunization of tumor-bearing mice with influenza virus, dual-specific T cells expanded greatly in numbers in the peritoneal cavity and spleen. This resulted in a significant increase in time of survival of mice. However, tumors were not eradicated, which may have been due to the observed poor penetration of tumor by T cells. This is the first demonstration that the potent immunogenic nature of an infectious agent can be utilized to directly impact on T-cell expansion and activity against tumor in vivo.

Cognition and motor control as a function of Delta9-THC concentration in serum and oral fluid: limits of impairment.

Cannabis use has been associated with increased risk of becoming involved in traffic accidents; however, the relation between THC concentration and driver impairment is relatively obscure. The present study was designed to define performance impairment as a function of THC in serum and oral fluid in order to provide a scientific framework to the development of per se limits for driving under the influence of cannabis. Twenty recreational users of cannabis participated in a double-blind, placebo-controlled, three-way cross-over study. Subjects were administered single doses of 0, 250 and 500 microg/kg THC by smoking. Performance tests measuring skills related to driving were conducted at regular intervals between 15 min and 6h post smoking and included measures of perceptual-motor control (Critical tracking task), motor impulsivity (Stop signal task) and cognitive function (Tower of London). Blood and oral fluid were collected throughout testing. Results showed a strong and linear relation between THC in serum and oral fluid. Linear relations between magnitude of performance impairment and THC in oral fluid and serum, however, were low. A more promising way to define threshold levels of impairment was found by comparing the proportion of observations showing impairment or no impairment as a function of THC concentration. The proportion of observations showing impairment progressively increased as a function of serum THC in every task. Binomial tests showed an initial and significant shift toward impairment in the Critical tracking task for serum THC concentrations between 2 and 5 ng/ml. At concentrations between 5 and 10 ng/ml approximately 75-90% of the observations were indicative of significant impairment in every performance test. At THC concentrations >30 ng/ml the proportion of observations indicative of significant impairment increased to a full 100% in every performance tests. It is concluded that serum THC concentrations between 2 and 5 ng/ml establish the lower and upper range of a THC limit for impairment.

Glucagon does not modulate the alterations in T3 metabolism consequent to dietary manipulation and diabetes.

Low serum T3 levels and hyperglucagonemia are characteristic features of a number of catabolic states such as fasting and uncontrolled diabetes. The present study was performed to elucidate the relationship between this hyperglucagonemia and T3 metabolism. Serum glucagon and T3 and hepatic T4-5'-deiodinase activity (T4 leads to T3) were examined in groups of rats (T4-treated) fed (chow versus carbohydrate), fasted, or diabetic (streptozotocin 100 mg/kg i.p.) for 48-72 h. In the carbohydrate-fed (20% glucose in H2O ad libitum) group the mean serum T3 concentration and mean hepatic T4-5'-deiodinase activity were significantly higher (P less than 0.01) and the mean serum glucagon level significantly lower (P less than 0.05) than the respective means in the chow-fed control group. The mean serum T3 concentration was significantly less (P less than 0.05) in both the fasted (72 h) and diabetic (72 h) groups compared with the control mean, whereas the mean serum glucagon values were similar to the chow-fed group. The mean hepatic T4-5'-deiodinase activity was low in the diabetic group (P less than 0.05) but similar in the fasted group compared with the chow-fed control. A significant inverse correlation (r = -0.9; P less than 0.001) was noted between these alterations in serum T3, hepatic T4-5'-deiodinase activity, and serum glucagon, suggesting that glucagon could be a modulator of T3 metabolism. Hyperglucagonemia was induced in the glucose-fed group with a continuous glucagon infusion for 48 h (0.15 micrograms/kg/min s.c.).(ABSTRACT TRUNCATED AT 250 WORDS)

Modulation of adipose lipoprotein lipase by thyroid hormone and diabetes. The significance of the low T3 state.

The present study was performed to assess the potential relationship between the low T3 syndrome and hypothyroidism. Comparative studies were performed on the relative effects of diabetes and insulin on heparin-releasable adipose lipoprotein lipase (LPL) in the intact and hypothyroid rat. Hypothyroidism for 10 days (Tx) significantly increased adipose LPL activity (5.8 +/- 0.2 mu eq/g/h) compared with the activity (3.6 +/- 0.4 mu eq/g/h) in the normal group. Diabetes for 72 h (streptozocin, STZ, 10 mg/100 g body wt, i.p.) significantly reduced (P less than 0.005) adipose LPL activity in the Tx model. However, despite the suppressant effect of diabetes (43 +/- 11%), the enzyme activity remained equivalent to the normal group. Insulin stimulated adipose LPL in the Tx-diabetic group. The enzyme demonstrated a synergistic response to insulin and hypothyroidism. Subsequent studies were performed in the intact diabetic rat, a low T3 state. Adipose LPL activity was reduced to a similar degree by diabetes (79 +/- 2%) irrespective of the serum T3 concentration. Furthermore, the magnitude of the adipose LPL stimulation by insulin was not modulated by the endogenous serum T3. However, co-treatment of the diabetic group with T3 and insulin blunted the adipose LPL response to insulin. These various modulations in adipose LPL activity were associated with significant but opposite changes in serum triglyceride levels in both the hypothyroid and intact rat. These studies demonstrate that hypothyroidism counteracts the suppressant effect of diabetes on heparin-releasable rat adipose LPL activity and magnifies the enzyme response to insulin.(ABSTRACT TRUNCATED AT 250 WORDS)

Trimethoprim-sulfamethoxazole therapy for Nocardia infections.

The optimal therapy for infections due to Nocardia species has not been established. To assess the efficacy of trimethoprim-sulfamethoxazole (TMP-SMX), we reviewed the records of 19 patients with Nocardia infections seen at Duke University Medical Center, Durham, NC, who were treated with this drug, either alone or in combination with other antibiotics or a surgical procedure. Underlying diseases or therapy causing immunosuppression were present in all but five cases. Sites of involvement were lung (ten of 19), wound (two of 19), and brain (two of 19); five of 19 patients had disseminated disease. The mean duration of therapy was 7.2 months. Overall cure or improvement was achieved in 89% (17/19) of cases; 80% of patients with disseminated disease and 60% of those with CNS involvement recovered. This experience, and accumulated clinical evidence in the literature, indicates that TMP-SMX should be considered the therapeutic drug of choice in infections due to Nocardia species.

Metabolic syndrome and aerobic fitness in patients with first-episode schizophrenia, including a 1-year follow-up.

OBJECTIVE: To compare the prevalence of metabolic syndrome (MetS) and metabolic abnormalities in patients with first-episode schizophrenia (FES) with sex- and age-matched healthy controls; to investigate changes in MetS during 1year of treatment; and to investigate predictors of MetS. METHODS: Patients with FES (N=99) and healthy controls (N=50) were included in the study. MetS was defined according to IDF based on waist circumference (WC), blood pressure (BP), triglycerides (TG), high-density lipoprotein (HDL), and fasting-glucose. Data on physical activity, aerobic fitness, smoking, and dietary habits, sleeping disturbances, psychopathology and psychotropic medication were also obtained. Patients were assessed at baseline and at 1year follow-up. RESULTS: Compared with healthy controls patients with FES had a higher baseline prevalence of MetS (p=.07), and metabolic abnormalities: WC (p<.01), TG (p<.01), HDL (p=.017), and fasting glucose (p=.04). Patients with FES had significantly increased prevalence of MetS (p=.03), WC (p=.04), and TG (p=.01) during the study period. Antipsychotics and low physical activity were significantly correlated with the increase in metabolic abnormalities. In multivariate analyses low aerobic fitness was the most consistent and significant predictor of metabolic abnormalities and MetS. CONCLUSION: MetS and metabolic abnormalities are highly prevalent in patients with FES, and both increase significantly during 1year of treatment. Apart from confirming the metabolic adverse effects of antipsychotics, our study highlights that low aerobic fitness is a significant risk factor for MetS. Promoting a healthier lifestyle should be part of psychiatric treatment and rehabilitation.

Polyglycidol-based metal adhesion promoters.

Hydrophilic adhesion promoters that facilitate intimate binding between metals and polymers are an important class of materials with a wide variety of applications in biomedical coatings. Currently, non-poly(meth-)acrylate based hydrophilic polymeric adhesives are unavailable. Here, we report the preparation of such adhesion-promoters based on linear polyglycidol for biomedical applications. The adhesion promoting polymer is prepared from partly phosphonoethylated polyglycidol in three steps. First, the remaining hydroxyl groups of the polyglycidol backbone are reacted with acryloyl chloride; secondly, the phosphonate groups are chemoselectively dealkylated using bromotrimethylsilane. Finally, the bis(trimethylsilyl)phosphonate intermediate is converted to the phosphonic acid through ethanolysis. The reaction conditions of each synthetic step are optimized individually and the products are characterized by 1H, 31P NMR and SEC analysis. The optimized reaction conditions are applied to establish a straightforward one-pot reaction, resulting in an ethanolic formulation of the adhesion promoter, which can be used immediately for the coating application. Special attention is paid to the stability of the intermediates, the chemoselectivity of the reactions and the shelf-life of the product. 1H NMR spectroscopy reveals hydrolytic instability of the product under ambient conditions; however, the polymers are sufficiently stable in dry ethanol for at least 14 days. The combination of this hydrophilic polymer with acrylate and phosphonic acid groups constitutes a versatile platform technology for the preparation of thin primer coatings on metal substrates for biomedical applications. The phosphonic acid residues assure strong binding to stainless steel wires and the acrylates can be addressed by UV light to enable crosslinking, thus improving mechanical stability and adhesion between the substrate and a biomedical hydrogel coating. The quality of the adhesion promotion to stainless steel wires is verified by using a lubricious, hydrogel top coat and by evaluating friction and wear resistance of this total coating system. Constant values for friction and wear are obtained, proving the applicability of phosphonic acid-functionalized polyglycidols as metal adhesion promoters for biomedical applications.

Therapeutic applications of viscous and injectable poly(ortho esters).

Poly(ortho esters) (POE) are hydrophobic and bioerodible polymers that have been investigated for pharmaceutical use since the early 1970s. Among the four described generations of POE, the third (POE III) and fourth (POE IV) are promising viscous and injectable materials which have been investigated in numerous biomedical applications. POE III has been extensively studied for ophthalmic drug delivery, it presents an excellent biocompatibility and is currently being investigated as a vehicle for sustained drug delivery to treat diseases of the posterior segment of the eye. POE IV is distinguishable by a highly reproducible and controlled synthesis, a higher hydrophobicity, and an excellent biocompatibility. It is currently under development for a variety of applications, such as ocular delivery, periodontal disease treatment and applications in veterinary medicine. This review will also focus on new perspectives for this promising family of polymers, such as guided tissue regeneration, treatment of osteoarthritis, as well as peptide and protein delivery.

Glucose and insulin reverse the effects of fasting on 3,5,3'-triiodothyronine neogenesis in primary cultures of rat hepatocytes.

The cellular mechanisms by which carbohydrate refeeding reverses the effect of fasting on T3 metabolism were studied in primary cultures of hepatocytes (24 h) harvested from 48-h fasted rats. Net T3 neogenesis (T3 generated from T4) in the fasted hepatocyte preparations (9.2 +/- 0.9 pmol/min X 100 mg protein) was significantly less (P less than 0.001) than that in hepatocyte cultures derived from 72-h glucose-fed rats (41 +/- 0.8 pmol/min X 100 mg protein). Preincubation (18 h) with either glucose (2.5-10 mM) or insulin (10-500 nM) significantly increased the fasted hepatocyte T3 levels to 28 +/- 0.6 and 22 +/- 1.3 pmol/min X 100 mg protein, respectively. Furthermore, incubation with both of these agents demonstrated a greater effect on hepatic T3 neogenesis than with either alone. Fasted hepatocyte T3 neogenesis was enhanced by enrichment with dithiothreitol (5 mM), but the T3 generation remained significantly less than that in cells exposed to glucose or insulin. Studies with glucose analogs demonstrated that preincubation with 2-deoxyglucose (5 mM) significantly increased (P less than 0.001) hepatocyte T3 neogenesis, but 3-O-methylglucose (5 mM) had no effect. In contrast, the insulin-mimetic compounds Concanavalin-A or spermine did not stimulate T3 neogenesis in the fasted hepatocyte cultures. Thus, rat hepatocytes sustained in primary culture for 24 h retain the T3 metabolic characteristics of the intact animal. Glucose and insulin reverse the effect of fasting on hepatocyte T3 neogenesis. The additive response to glucose and insulin suggests that T3 neogenesis is modulated through different mechanisms. The replication of the glucose effect by 2-deoxyglucose and the inability of dithiothreitol to reverse the effect of fasting on hepatocyte T4 5'-deiodinase activity suggest that neither intermediates in the glycolytic pathway nor thiol cofactors mediate the glucose effect. Thus, the restoration of liver T3 metabolism consequent to carbohydrate refeeding of the fasted rat may be mediated by the glucose and insulin responses.

Carbohydrate feeding increases total body and specific tissue 3,5,3'-triiodothyronine neogenesis in the rat.

The glucose-fed rat, in contrast to the chow-fed animal, has a higher serum total T3 concentration and an increase in the hepatic content of T4 5'-deiodinase (type I) activity. The mechanism and significance of these glucose-induced changes in T3 metabolism are elucidated in this study. To focus on extrathyroidal thyroid hormone metabolism the kinetic parameters were determined in thyroidectomized T4-replaced rats (1.25 micrograms T4/100 g BW.day). Kinetics of T4 and T3 were studied separately by infusing labeled hormone to equilibrium. Glucose feeding for 72 h (G) significantly increased both the total and free serum T3 concentrations compared to the respective means in the chow-fed control group (P). The glucose-induced changes in serum T3 reflect the approximate doubling of T3 production to 14.7 +/- 0.6 ng/h.100 g in G rats compared to 7.6 +/- 0.7 ng/h.100 g in P rats. The higher T3 production rate in the G group is due to a significant increase in the fractional total body T4 to T3 conversion (0.33 +/- 0.02) compared to that in the P group (0.19 +/- 0.02). The tissue (liver, kidney, brain, and brown adipose tissue) concentration of T4 (nanograms per g wet wt) was significantly increased in the G group. The increase ranged from 54% in liver to 80% in kidney, brain, and brown adipose tissue. The tissue concentration of T3 (nanograms per g wet wt) was even more dramatically increased by glucose feeding than was T4. The glucose-induced increment in organ T3 ranged from 2.5-fold (kidney, muscle, and brain) to 5-fold (liver and white adipose tissue) to 12-fold (brown adipose tissue). These data indicate that the increase in serum total and free T3 concentrations associated with glucose feeding reflects augmented total body T3 production from T4. The effect of the enhanced T3 neogenesis was generalized, as the T3 content was increased in each organ studied. Thus, glucose feeding has unique effects on T3 metabolism.

Molecular Cloning and Characterization of c-erb-A mRNA Species in Mouse Neuroblastoma Cells.

UNLABELLED: Abstract The mouse neuroblastoma cell line is an excellent model in which to study thyroid hormone action and metabolism, particularly in neural tissue. We therefore undertook the molecular cloning and characterization in these cells of putative thyroid hormone receptors related to c-erb-A. Since rat brain tissue contains multiple cell types, and because of possible subtle differences between species (mouse and rat), we therefore screened a new cDNA library constructed from mouse neuroblastoma cell mRNA with synthetic oligonucleotide probes based on the published nucleotide sequence of the c-erb-A gene in whole rat brain. Despite the fact that this rat brain cDNA sequence is now recognized to represent a c-erb-A alpha 1 form, the cDNA clones that we isolated were all members of the newly-recognized c-erb-A alpha 2 form. This identification was made on the basis of nucleotide sequence divergence downstream of the nucleotide corresponding to amino-acid residue 370 in the predicted coding region. The two longest mouse neuroblastoma cDNA clones, clone 29 (1796 bp) and clone 32 (1410 bp), were 93% to 94% homologous with the c-erb-A alpha 2 and c-erb-A alpha 1 forms in their DNA binding and thyroid hormone binding domains (up to amino-acid residue 370 in the latter). Both clones 29 and 32 were incomplete in that they terminated at their 3'ends at an internal Eco R1 site. Fortunately this 120 bp (40 derived amino-acid) truncation was downstream of the reported thyroid hormone binding domain. The 5'untranslated end of clone 29 (446 bp) was of interest because a region of its nucleotide sequence (279 bp) revealed a high degree of homology (87%) with rat brain c-erb-A alpha 1. This highly conserved region in clone 29 appeared to be important in the regulation of translation because only clone 32, in which this region was truncated, efficiently translated protein in a cell-free system. The protein product of clone 32 did not bind thyroid hormone. Northern blot analysis of mouse neuroblastoma mRNA with site-specific synthetic oligonucleotides revealed that the c-erb-A alpha 2 species was dominant (major band of 2.4 kb), with a lesser amount of the c-erb-A alpha1 species (major band of 1.8 kb). IN CONCLUSION: 1) We report the molecular cloning of c-erb-A variants in a specific neural tissue cell type, namely mouse neuroblastoma cells; 2) The cells contained predominantly the c-erb-A alpha 2 subtype; 3) This c-erb-A alpha 2 form was unique up to bp 167 at its 5'untranslated end, and was then followed, up to the ATG initiation codon, by a highly conserved region common to all c-erb-A a species; 4) The 5'untranslated region appeared to play a role in the translational efficiency of this mRNA; 5) The protein product of the c-erb-A alpha 2 mRNA in these cells did not appear to bind thyroid hormone. In view of this finding, the physiological role of the c-erb-A alpha 2 protein remained speculative and may involve a represser function at the thyroid hormone responsive element.